Soybean isopentenyl transferase genes and methods of use

ABSTRACT

Methods and compositions for modulating plant development are provided. Polynucleotide sequences encoding isopentenyl transferase (IPT) polypeptides are provided, as are the amino acid sequences of the encoded polypeptides. The sequences can be used in a variety of methods including modulating root development, modulating floral development, modulating leaf and/or shoot development, modulating senescence, modulating seed size and/or weight, and modulating tolerance of plants to abiotic stress. Transformed plants, plant cells, tissues, and seed are also provided.

CROSS REFERENCE

This utility application claims the benefit U.S. Provisional ApplicationNo. 60/764,303, filed Feb. 1, 2006, which is incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates to the field of genetic manipulation of plants,particularly the modulation of gene activity to affect plant developmentand growth.

BACKGROUND OF THE INVENTION

Cytokinins are a class of N⁶ substituted purine derivative planthormones that regulate cell division and influence a large number ofdevelopmental events, such as shoot development, sink strength, rootbranching, control of apical dominance in the shoot, leaf development,chloroplast development, and leaf senescence (Mok, et al., (1994)Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, Fla.,pp. 155-166; Horgan (1984) Advanced Plant Physiology ed. MB., Pitman,London, UK, pp 53-75; and Letham (1994) Annual Review of Plant Physiol34:163-197). In maize, cytokinins (CK) play an important role inestablishing seed size, decreasing tip kernel abortion, and increasingseed set during unfavorable environmental conditions (Cheikh, et al.,(1994) Plant Physiol. 106:45-51; Dietrich, et al., (1995) Plant PhysiolBiochem 33:327-36). Active cytokinin pools are regulated by rates ofsynthesis and degradation.

Until recently, roots were believed to be the major site of cytokininbiosynthesis but evidence indicates that others tissues, such as shootmeristems and developing seeds, also have high cytokinin biosyntheticactivity. It has been suggested that cytokinins are synthesized inrestricted sites where cell proliferation is active. The presence ofseveral AtIPT genes in Arabidopsis and their differential pattern ofexpression might serve this purpose.

The catabolic enzyme isopentenyl transferase (IPT) directs the synthesisof cytokinins and plays a major role in controlling cytokinin levels inplant tissues. Multiple routes have been proposed for cytokininbiosynthesis. Transfer RNA degradation has been suggested to be a sourceof cytokinin, because some tRNA molecules contain anisopentenyladenosine (iPA) residue at the site adjacent to the anticodon(Swaminathan, et al., (1977) Biochemistry 16:1355-1360). Themodification is catalyzed by tRNA isopentenyl transferase (tRNA IPT; EC2.5.1.8), which has been identified in various organisms such asEscherichia coli, Saccharomyces cerevisiae, Lactobacillus acidophilus,Homo sapiens, and Zea mays (Bartz, et al., (1972) Biochemie 54:31-39;Kline, et al., (1969) Biochemistry 8:4361-4371; Holtz, et al., (1975)Hoppe-Seyler's Z. Physiol. Chem. 356:1459-1464; Golovko, et al., (2000)Gene 258:85-93; and, Holtz, et al., (1979) Hoppe-Seyler's Z. Physiol.Chem. 359:89-101). However, this pathway is not considered to be themain route for cytokinin synthesis (Chen, et al., (1997) Physiol. Plant101:665-673 and McGraw, et al., (1995) Plant Hormones, Physiology,Biochemistry and Molecular Biology. Ed. Davies, 98-117, Kluwer AcademicPublishers, Dordrecht).

Another possible route of cytokinin formation is de novo biosynthesis ofiPMP by adenylate isopentenyl transferase (IPT; EC 2.5.1.27) withdimethylallyl-diphosphate (DMAPP), AMP, ATP, and ADP as substrates. Ourcurrent knowledge of cytokinin biosynthesis in plants is largely deducedfrom studies on a possible analogous system in Agrobacteriumtumefaciens. Cells of A. tumefaciens are able to infect certain plantspecies by inducing tumor formation in host plant tissues (Van Montagu,et al., (1982) Curr Top Microbiol Immunol 96:237-254; Hansen, et al.,(1999). Curr Top Microbiol Immunol 240:21-57). To do so, the A.tumefaciens cells synthesize and secrete cytokinins which mediate thetransformation of normal host plant tissues into tumors or calli. Thisprocess is facilitated by the A. tumefaciens tumor-inducing plasmidwhich contains genes encoding the necessary enzyme and regulators forcytokinin biosynthesis. Biochemical and genetic studies revealed thatGene 4 of the tumor-inducing plasmid encodes an isopentenyl transferase(IPT), which converts AMP and DMAPP intoisopentenyladenosine-5′-monophosphate (iPMP), the active form ofcytokinins (Akiyoshi, et al., (1984) Proc. Natl. Acad. Sci. USA81:5994-5998). Overexpression of the Agrobacterium ipt gene in a varietyof transgenic plants has been shown to cause an increased level ofcytokinins and elicit typical cytokinin responses in the host plant(Hansen, et al., (1999) Curr Top Microbiol Immunol 240:21-57).Therefore, it has been postulated that plant cells use machinery similarto that of A. tumefaciens cells for cytokinin biosynthesis. ArabidopsisIPT homologs have recently been identified in Arabidopsis and Petunia(Takei, et al., (2001) J. Biol. Chem. 276:26405-26410 and Kakimoto(2001) Plant Cell Physiol. 42:677-685). Overexpression of theArabidopsis IPT homologs in plants elevated cytokinin levels andelicited typical cytokinin responses in planta and under tissue cultureconditions (Kakimoto (2001) Plant Cell Physiol. 42:677-685).

Arabidopsis ipt genes are members of a small multigene family of ninedifferent genes, two of which code for tRNA isopentenyl transferases,and seven of which encode a gene product with a cytokinin biosyntheticfunction. Biochemical analysis of the recombinant AtIPT4 protein showedthat, in contrast to the bacterial enzyme, the Arabidopsis enzyme usesATP as a substrate instead of AMP. Another plant IPT gene (Sho) wasidentified in Petunia hybrida using an activation tagging strategy(Zubko, et al., (2002) The Plant Journal 29:797-808).

In view of the influence of cytokinins on a wide variety of plantdevelopmental processes, including root architecture, shoot and leafdevelopment, and seed set, the ability to manipulate cytokinin levels inhigher plant cells, and thereby drastically effect plant growth andproductivity, offers significant commercial value (Mok, et al., (1994)Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, Fla.,pp. 155-166).

BRIEF SUMMARY OF THE INVENTION

Compositions and methods of the invention comprise and employisopentenyl transferase (IPT) polypeptides and polynucleotides that areinvolved in modulating plant development, morphology, and physiology.

Compositions further include expression cassettes, plants, plant cells,and seeds having the IPT sequences of the invention. The plants, plantcells, and seeds of the invention may exhibit phenotypic changes, suchas modulated (increased or decreased) cytokinin levels; modulated floraldevelopment; modulated root development; altered shoot to root ratio;increased seed size or an increased seed weight; increased plant yieldor plant vigor; maintained or improved stress tolerance (e.g., increasedor maintained size of the plant, minimized seed or pod abortion,increased or maintained seed set); decreased shoot growth; delayedsenescence or an enhanced vegetative growth, all relative to a plant,plant cell, or seed not modified per the invention.

Methods are provided for reducing or eliminating the activity of an IPTpolypeptide in a plant, comprising introducing into the plant a selectedpolynucleotide. In specific methods, providing the polynucleotidedecreases the level of cytokinin in the plant and/or modulates rootdevelopment of the plant.

Methods are also provided for increasing the level of an IPT polypeptidein a plant comprising introducing into the plant a selectedpolynucleotide. In specific methods, expression of the IPTpolynucleotide increases the level of a cytokinin in the plant;maintains or improves the stress tolerance of the plant; maintains orincreases the size of the plant; minimizes seed abortion; increases ormaintains seed set; increases shoot growth; increases seed size or seedweight; increases plant yield or plant vigor; modulates floraldevelopment; delays senescence; or increases leaf growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an alignment of amino acid sequences of ZmIPT2 (SEQ IDNO: 8), GmIPT1 (SEQ ID NO: 2), GmIPT2 (SEQ ID NO: 4), and GmIPT3 (SEQ IDNO: 7). Asterisks indicate amino acids conserved in many IPT proteins,and the derived IPT consensus sequence is set out below the alignment. Amotif characteristic of tRNA IPT was found in GmIPT3.

FIG. 2 provides percent identity and percent similarity values forGmIPT1, GmIPT2, GmIPT3, ZmIPT2, and Arabidopsis IPT1-IPT9.

FIG. 3 is a Northern blot showing relative levels of expression ofGmIPT1 (Panel A) and GmIPT2 (Panel B) in various soybean tissues.

FIG. 4 is a phylogenetic tree of plant IPT sequences. GmIPT1 and GmIPT2are clustered with other plant IPT proteins whereas GmIPT3 clusters withAtIPT2 which is a tRNA IPT.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 and 2 provide nucleotide and amino acid sequences forGmIPT1.

SEQ ID NO: 3 and 4 provide nucleotide and amino acid sequences forGmIPT2.

SEQ ID NO: 5 provides the full insert sequence for the GmIPT2 ESTinitially identified.

SEQ ID NO: 6 and 7 provide nucleotide and amino acid sequences forGmIPT3.

SEQ ID NO: 8 provides the ZmIPT2 amino acid sequence.

SEQ ID NO: 9 is a consensus IPT sequence.

SEQ ID NOS: 10-13 are primer sequences used in BAC screening.

SEQ ID NOS: 14-16 are tags used in expression profiling.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not all,embodiments of the invention are shown. Indeed, the invention may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the invention set forthherein will come to mind to one skilled in the art to which thisinvention pertains having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

Compositions

Compositions of the invention include isopentenyl transferase (IPT)polypeptides and polynucleotides that are involved in modulating plantdevelopment, morphology, and physiology. In particular, the presentinvention provides for isolated polynucleotides comprising nucleotidesequences encoding the amino acid sequences shown in SEQ ID NO: 2, 4,and 7. Further provided are isolated polypeptides having an amino acidsequence encoded by a polynucleotide described herein, for example thoseset forth in SEQ ID NO: 1, 3, and 6.

The isopentenyl transferase polypeptides of the invention share sequenceidentity with members of the isopentenyl transferase family of proteins.Polypeptides in the IPT family have been identified in various bacteriaand in Arabidopsis and Petunia. See, for example, Kakimoto (2001) PlantCell Physio. 42:677-658; Takei, et al., (2001) The Journal of BiologicalChemistry 276:26405-26410; and Zubko, et al., (2002) The Plant Journal29:797-808. Members of the IPT family are characterized by having theconsensus sequence GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG [ST] (SEQ IDNO: 9) (where x denotes any amino acid residue, [ ] any one of the aminoacids shown in [ ], and x{m,n} m to n amino acid residues in number).See, Kakimoto, et al., (2001) Plant Cell Physiol. 42:677-85 andKakimoto, et al., (2003) J. Plant Res. 116:233-9, both of which areherein incorporated by reference. IPT family members may also haveATP/GTP binding sites. An amino acid alignment of the maize IPT2 proteinalong with soy (Glycine max) cytokinin biosynthetic enzymes of theinvention is provided in FIG. 1. Asterisks indicate a consensus sequencefound in many cytokinin biosynthetic enzymes. In addition to thisconsensus sequence, a tRNA binding site was identified in GmIPT3 whichsuggests that the gene encodes a tRNA IPT enzyme.

Isopentenyl transferase enzymes are involved in cytokinin biosynthesis,therefore the IPT polypeptides of the invention have “cytokininsynthesis activity.” By “cytokinin synthesis activity” is intendedenzymatic activity that generates cytokinins, derivatives thereof, orany intermediates in the cytokinin synthesis pathway. Cytokininsynthesis activity therefore includes, but is not limited to, DMAPP:AMPisopentenyltransferase activity (the conversion of AMP(adenosine-5′-monophosphate) and DMAPP into iPMP(isopentenyladenosine-5′-monophosphate)), DMAPP:ADPisopentenyltransferase activity (the conversion of ADP(adenosine-5′-diphosphate) and DMAPP into iPDP(isopentenyladenosine-5′-diphosphate)); DMAPP:ATP isopentenyltransferaseactivity (the conversion of ATP (adenosine-5′-triphosphate) and DMAPPinto iPTP (isopentenyladenosine-5′-triphosphate)), and DMAPP:tRNAisopentenyltransferase activity (the modification of cytoplasmic,chloroplastic and/or mitochondrial tRNAs to give isopentenyl). Cytokininsynthesis activity can further include a substrate comprising a secondside chain precursor, other than DMAPP. Examples of side chain donorsinclude compounds of terpenoid origin. For example, the substrate couldbe hydroxymethylbutenyl diphosphate (HMBPP) which would allowtrans-zeatin riboside monophosphate (ZMP) synthesis. See, for example,Åstot, et al., (2000) Proc Natl Acad Sci 97:14778-14783 and Takei, etal., (2003) J Plant Res. 116(3):265-9.

Cytokinin synthesis activity further includes the synthesis ofintermediates involved in formation of ZMP. Methods to assay for theproduction of various cytokinins and their intermediates can be found,for example, in Takei, et al., (2001) The Journal of BiologicalChemistry 276:26405-26410, Zubo, et al., (2002) The Plant Journal29:797-808; Kakimoto, et al., (2001) Plant Cell Physio. 42:677-658, andSun, et al., (2003) Plant Physiology 131:167-176, each of which isherein incorporated by reference. “Cytokinin synthesis activity” alsoincludes any alteration in a plant or plant cell phenotype that ischaracteristic of an increase in cytokinin concentration. Such cytokininspecific effects are discussed elsewhere herein and include, but are notlimited to, enhanced shoot formation, reduced apical dominance, delayedsenescence, delayed flowering, increased leaf growth, increasedcytokinin levels in the plant, increased tolerance under stress,minimization of pod and/or seed abortion, increased or maintained seedset under stress conditions, and a decrease in root growth. Assays tomeasure or detect such phenotypes are known. See, for example, Miyawaki,et al., (2004) The Plant Journal 37:128-138, Takei, et al., (2001) TheJournal of Biological Chemistry 276:26405-26410, Zubo, et al., (2002)The Plant Journal 29:797-808; Kakimoto, et al., (2001) Plant CellPhysio. 42:677-658, and Sun, et al., (2003) Plant Physiology131:167-176, each of which is herein incorporated by reference.Additional phenotypes resulting from an increase in cytokinin synthesisactivity in a plant are discussed herein.

Compositions of the invention include IPT sequences that are involved incytokinin biosynthesis. In particular, the present invention providesfor isolated polynucleotides comprising nucleotide sequences encodingthe amino acid sequences shown in SEQ ID NO: 2, 4 and 7. Furtherprovided are polypeptides having an amino acid sequence encoded by apolynucleotide described herein, for example those set forth in SEQ IDNOS: 1, 3 and 6, and fragments and variants thereof.

The invention encompasses isolated or substantially purifiedpolynucleotide or protein compositions. An “isolated” or “purified”polynucleotide or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Fragments and variants of the disclosed polynucleotides and proteinsencoded thereby are also encompassed by the present invention. By“fragment” is intended a portion of the polynucleotide or a portion ofthe amino acid sequence and hence protein encoded thereby. Fragments ofa polynucleotide may encode protein fragments that retain the biologicalactivity of the native protein and hence have cytokinin synthesisactivity. Alternatively, fragments of a polynucleotide that are usefulas hybridization probes generally do not encode fragment proteinsretaining biological activity. Thus, fragments of a nucleotide sequencemay range from at least about 20 nucleotides, about 50 nucleotides,about 100 nucleotides, and up to the full-length polynucleotide encodingthe proteins of the invention.

A fragment of an IPT polynucleotide that encodes a biologically activeportion of an IPT protein of the invention will encode at least 15, 25,30, 50, 100, 150, 200, 225, 250, 275, 300, 310, 315 or 320 contiguousamino acids, or up to the total number of amino acids present in afull-length IPT protein of the invention (for example, 340 amino acidsfor SEQ ID NO: 2 or 4; 480 amino acids for SEQ ID NO: 7). Fragments ofan IPT polynucleotide that are useful as hybridization probes or PCRprimers generally need not encode a biologically active portion of anIPT protein.

Thus, a fragment of an IPT polynucleotide may encode a biologicallyactive portion of an IPT protein, or it may be a fragment that can beused as a hybridization probe or PCR primer using methods disclosedbelow. A biologically active portion of an IPT protein can be preparedby isolating a portion of one of the IPT polynucleotides of theinvention, expressing the encoded portion of the IPT protein (e.g., byrecombinant expression in vitro), and assessing the activity of theencoded portion of the IPT protein. Polynucleotides that are fragmentsof an IPT nucleotide sequence comprise at least 16, 20, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900,950 or 965 contiguous nucleotides, or up to the number of nucleotidespresent in a full-length IPT polynucleotide disclosed herein (forexample, 1023, 1409, and 1592 nucleotides for SEQ ID NO: 1, 3 and 6,respectively).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. For polynucleotides,conservative variants include those sequences that, because of thedegeneracy of the genetic code, encode the amino acid sequence of one ofthe IPT polypeptides of the invention. Naturally occurring variants suchas these can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR) andhybridization techniques as outlined below. Variant polynucleotides alsoinclude synthetically derived polynucleotide, such as those generated,for example, by using site-directed mutagenesis but which still encodean IPT protein of the invention. Generally, variants of a particularpolynucleotide of the invention will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to that particularpolynucleotide as determined by sequence alignment programs andparameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, isolated polynucleotides that encodea polypeptide with a given percent sequence identity to the polypeptideof SEQ ID NO: 2, 4 or 7 are disclosed. Percent sequence identity betweenany two polypeptides can be calculated using sequence alignment programsand parameters described elsewhere herein. Where any given pair ofpolynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore sites in the native protein and/or substitution of one or moreamino acids at one or more sites in the native protein. Certain variantproteins encompassed by the present invention are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, cytokinin synthesis activity, as describedherein. Such variants may result from, for example, genetic polymorphismor from human manipulation. Biologically active variants of a native IPTprotein of the invention will have at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to the amino acid sequence for thenative protein as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa protein of the invention may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2 or even 1 amino acid residue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the IPT proteinscan be prepared by mutations in the DNA. Methods for mutagenesis andpolynucleotide alterations are well known in the art. See, for example,Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel, et al.,(1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walkerand Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model ofDayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl.Biomed. Res. Found., Washington, D.C.), herein incorporated byreference. Conservative substitutions, such as exchanging one amino acidwith another having similar properties, may be optimal.

Thus, the genes and polynucleotides of the invention include both thenaturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass naturally occurring proteins as wellas variations and modified forms thereof. Such variants will continue topossess the desired IPT activity. Obviously, the mutations that will bemade in the DNA encoding the variant must not place the sequence out ofreading frame and optimally will not create complementary regions thatcould produce secondary mRNA structure.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by assaying for cytokinin synthesis activity. See, forexample, Takei, et al., (2001) The Journal of Biological Chemistry276:26405-26410; Zubo, et al., (2002) The Plant Journal 29:797-808;Kakimoto, et al., (2001) Plant Cell Physio. 42:677-658; Sun, et al.,(2003) Plant Physiology 131:167-176; and Miyawaki, et al., (2004) ThePlant Journal 37:128-138, all of which are herein incorporated byreference.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different IPT codingsequences can be manipulated to create a new IPT polypeptide possessingthe desired properties. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. For example, using this approach, sequence motifs encoding adomain of interest may be shuffled between the IPT gene of the inventionand other known IPT genes to obtain a new gene coding for a protein withan improved property of interest, such as an increased K_(m) in the caseof an enzyme. Strategies for such DNA shuffling are known in the art.See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri, et al.,(1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; PCTpublication WO97/20078; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

By “promoter” is intended a regulatory region of DNA usually comprisinga TATA box capable of directing RNA polymerase II to initiate RNAsynthesis at the appropriate transcription initiation site for aparticular polynucleotide sequence. A promoter may additionally compriseother recognition sequences generally positioned upstream or 5′ to theTATA box, referred to as upstream promoter elements, which influence thetranscription initiation rate. The promoter sequences of the presentinvention regulate (i.e., repress or activate) transcription.

The polynucleotides of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire IPT sequencesset forth herein or to variants and fragments thereof are encompassed bythe present invention. Such sequences include sequences that areorthologs of the disclosed sequences. “Orthologs” is intended to meangenes derived from a common ancestral gene and which are found indifferent species as a result of speciation. Genes found in differentspecies are considered orthologs when their nucleotide sequences and/ortheir encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequenceidentity. Functions of orthologs are often highly conserved amongspecies. Thus, isolated polynucleotides that encode an IPT protein andwhich hybridize under stringent conditions to the IPT sequencesdisclosed herein, or to variants or fragments or complements thereof,are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methodsand Applications (Academic Press, New York); Innis and Gelfand, eds.(1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand,eds. (1999) PCR Methods Manual (Academic Press, New York). Known methodsof PCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker.Thus, for example, probes for hybridization can be made by labelingsynthetic oligonucleotides based on the IPT polynucleotides of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

For example, an entire IPT polynucleotide disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding IPT polynucleotides. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique among IPT polynucleotide sequences and areoptimally at least about 10 nucleotides in length, and most optimally atleast about 20 nucleotides in length. Such probes may be used to amplifycorresponding IPT polynucleotides from a chosen plant by PCR. Thistechnique may be used to isolate additional coding sequences from adesired plant or as a diagnostic assay to determine the presence ofcoding sequences in a plant. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook, et al., (1989) Molecular Cloning:A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is optimal to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel, et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See, Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith, et al., (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins, et al.,(1988) Gene 73:237-244 (1988); Higgins, et al., (1989) CABIOS 5:151-153;Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al.,(1992) CABIOS 8:155-65; and Pearson, et al., (1994) Meth. Mol. Biol.24:307-331. The ALIGN program is based on the algorithm of Myers andMiller (1988) supra. A PAM120 weight residue table, a gap length penaltyof 12, and a gap penalty of 4 can be used with the ALIGN program whencomparing amino acid sequences. The BLAST programs of Altschul, et al.,(1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin andAltschul (1990) supra. BLAST nucleotide searches can be performed withthe BLASTN program, score=100, wordlength=12, to obtain nucleotidesequences homologous to a nucleotide sequence encoding a protein of theinvention. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul, et al., (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See, Altschul, et al., (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See, www.ncbi.nlm.nih.gov. Alignment may also be performedmanually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see, Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The invention further provides plants having altered levels and/oractivities of the IPT polypeptides of the invention. In someembodiments, the plants of the invention have stably incorporated intotheir genome the IPT sequences of the invention. In other embodiments,plants that are genetically modified at a genomic locus encoding an IPTpolypeptide of the invention are provided. By “native genomic locus” isintended a naturally occurring genomic sequence. The genomic locus maybe modified to reduce or eliminate the activity of the IPT polypeptide.The term “genetically modified” as used herein refers to a plant orplant part that is modified in its genetic information by theintroduction of one or more foreign polynucleotides, and the insertionof the foreign polynucleotide leads to a phenotypic change in the plant.By “phenotypic change” is intended a measurable change in one or morecell functions. For example, plants having a genetic modification at thegenomic locus encoding the IPT polypeptide can show reduced oreliminated expression or activity of the IPT polypeptide. Variousmethods to generate such a genetically modified genomic locus aredescribed elsewhere herein, as are the variety of phenotypes that canresult from the modulation of the level/activity of the IPT sequences ofthe invention.

As used herein, the term plant includes reference to whole plants, plantparts or organs (e.g., leaves, stems, roots), plant cells, and seeds andprogeny of same. Plant cell, as used herein, includes, withoutlimitation, cells obtained from or found in seeds, suspension cultures,embryos, meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores, as well as plantprotoplasts and plant cell tissue cultures, plant calli, plant clumps,and plant cells that are intact in plants or parts of plants such asembryos, pollen, ovules, seeds, leaves, flowers, branches, fruit,kernels, ears, cobs, husks, stalks, roots, root tips, anthers, grain andthe like. As used herein, “grain” refers to the mature seed produced bycommercial growers for purposes other than growing or reproducing thespecies. Progeny, variants, and mutants of the regenerated plants arealso included within the scope of the invention, provided that theseparts comprise the introduced nucleic acid sequences.

Methods

I. Providing Sequences

The sequences of the present invention can be introduced into andexpressed in a host cell such as bacteria, yeast, insect, mammalian, oroptimally plant cells. It is expected that those of skill in the art areknowledgeable in the numerous systems available for the introduction ofa polypeptide or a nucleotide sequence of the present invention into ahost cell. No attempt to describe in detail the various methods knownfor providing proteins in prokaryotes or eukaryotes will be made.

By “host cell” is meant a cell which comprises a heterologous nucleicacid sequence of the invention. Host cells may be prokaryotic cells suchas E. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells. Host cells can also be monocotyledonous ordicotyledonous plant cells. In certain embodiments, the monocotyledonoushost cell is a maize host cell.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

The IPT polynucleotides of the invention can be provided in expressioncassettes for expression in the plant of interest. The cassette willinclude 5′ and 3′ regulatory sequences operably linked to an IPTpolynucleotide of the invention. “Operably linked” is intended to mean afunctional linkage between two or more elements. For example, anoperable linkage between a polynucleotide of interest and a regulatorysequence (i.e., a promoter) is a functional link that allows forexpression of the polynucleotide of interest. Operably linked elementsmay be contiguous or non-contiguous. When used to refer to the joiningof two protein coding regions, by operably linked is intended that thecoding regions are in the same reading frame. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes. An expression cassette may be providedwith a plurality of restriction sites and/or recombination sites forinsertion of the IPT polynucleotide to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain selectable marker genes.

In certain embodiments, the expression cassette will include in the5′-3′ direction of transcription, a transcriptional and translationalinitiation region (i.e., a promoter), an IPT polynucleotide of theinvention, and a transcriptional and translational termination region(i.e., termination region) functional in plants. The regulatory regions(i.e., promoters, transcriptional regulatory regions, and translationaltermination regions) and/or the IPT polynucleotide of the invention maybe native/analogous to the host cell or to each other. Alternatively,the regulatory regions and/or the IPT polynucleotide of the inventionmay be heterologous to the host cell or to each other. As used herein,“heterologous” in reference to a sequence is a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention. For example, a promoter operably linkedto a heterologous polynucleotide is from a species different from thespecies from which the polynucleotide was derived, or, if from thesame/analogous species, one or both are substantially modified fromtheir original form and/or genomic locus, or the promoter is not thenative promoter for the operably-linked polynucleotide. As used herein,a chimeric gene comprises a coding sequence operably linked to atranscription initiation region that is heterologous to the codingsequence.

While heterologous promoters can be used to express the IPT sequences,the native promoter sequences or other IPT promoters may also be used.Such constructs can change expression levels of IPT sequences in theplant or plant cell. Thus, the phenotype of the plant or plant cell canbe altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably-linked IPT polynucleotide ofinterest, may be native with the plant host, or may be derived fromanother source (i.e., foreign or heterologous with reference to thepromoter), the IPT polynucleotide of interest, the plant host, or anycombination thereof. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also, Guerineau, et al.,(1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674;Sanfacon, et al., (1991) Genes Dev. 5:141-149; Mogen, et al., (1990)Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas,et al., (1989) Nucleic Acids Res. 17:7891-7903; and Joshi, et al.,(1987) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed plant. That is, the polynucleotides can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray, et al., (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154:9-20), and human immunoglobulin heavy-chain bindingprotein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virusleader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed.Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virusleader (MCMV) (Lommel, et al., (1991) Virology 81:382-385). See also,Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968. Other methodsknown to enhance translation can also be utilized.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su, et al., (2004)Biotechnol Bioeng 85:610-9 and Fetter, et al., (2004) Plant Cell16:215-28), cyan fluorescent protein (CYP) (Bolte, et al., (2004) J.Cell Science 117:943-54 and Kato, et al., (2002) Plant Physiol129:913-42), and yellow fluorescent protein (PhiYFP™ from Evrogen, see,Bolte, et al., (2004) J. Cell Science 117:943-54). For additionalselectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech.3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff (1992) Mol.Microbiol. 6:2419-2422; Barkley, et al., (1980) in The Operon, pp.177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al., (1987) Cell49:603-612; Figge, et al., (1988) Cell 52:713-722; Deuschle, et al.,(1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst, et al., (1989)Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990)Science 248:480-483; Gossen (1993) Ph.D. Thesis, University ofHeidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol. 10:3343-3356;Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim,et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, etal., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989)Topics Mol. Struc. Biol. 10:143-162; Degenkolb, et al., (1991)Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988)Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University ofHeidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka, et al., (1985) Handbook of ExperimentalPharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill, et al., (1988)Nature 334:721-724. Such disclosures are herein incorporated byreference. The above list of selectable marker genes is not meant to belimiting. Any selectable marker gene can be used in the presentinvention.

A number of promoters can be used in the practice of the invention,including the native promoter of the polynucleotide sequence ofinterest. The promoters can be selected based on the desired outcome.The nucleic acids can be combined with constitutive, inducible,tissue-preferred, or other promoters for expression in plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell,et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990)Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol.Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol.18:675-689); PEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S.Pat. No. 5,659,026), and the like. Other constitutive promoters include,for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to target enhanced IPTexpression within a particular plant tissue. Tissue-preferred promotersinclude Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kawamata, etal., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997)Mol. Gen. Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res.6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341;Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, etal., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994)Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. CellDiffer. 20:181-196; Orozco, et al., (1993) Plant Mol. Biol.23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia, et al., (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression. See, also, U.S. Patent Application No. 2003/0074698, hereinincorporated by reference.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138; Baszczynski, et al., (1988)Nucl. Acid Res. 16:4732; Mitra, et al., (1994) Plant Molecular Biology26:35-93; Kayaya, et al., (1995) Molecular and General Genetics248:668-674; and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA90(20):9586-9590. Senecence regulated promoters are also of use, suchas, SAM22 (Crowell, et al., (1992) Plant Mol. Biol. 18:459-466). See,also, U.S. Pat. No. 5,589,052 herein incorporated by reference.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire, et al., (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger, et al.,(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao,et al., (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed roIC and roID root-inducinggenes of Agrobacterium rhizogenes (see. Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri, et al., (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see, EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772); roIBpromoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691; and theCRWAQ81 root-preferred promoter with the ADH first intron (U.S. PatentPublication 2005/0097633). See also, U.S. Pat. Nos. 5,837,876;5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

“Seed-preferred” promoters refers to those promoters active during seeddevelopment and may include expression in seed initials or relatedmaternal tissue. Such seed-preferred promoters include, but are notlimited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDazein); milps (myo-inositol-1-phosphate synthase) (see, WO 00/11177 andU.S. Pat. No. 6,225,529; herein incorporated by reference). Gamma-zeinis an endosperm-specific promoter. Globulin-1 (Glob-1) is arepresentative embryo-specific promoter. For dicots, seed-specificpromoters include, but are not limited to, bean β-phaseolin, napin,β-conglycinin, soybean lectin, cruciferin, and the like. For monocots,seed-specific promoters include, but are not limited to, maize 15 kDazein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken2, globulin 1, etc. See also, WO 00/12733, where seed-preferredpromoters from end1 and end2 genes are disclosed; herein incorporated byreference. Additional embryo specific promoters are disclosed in Sato,et al., (1996) Proc. Natl. Acad. Sci. 93:8117-8122; Nakase, et al.,(1997) Plant J 12:235-46; and Postma-Haarsma, et al., (1999) Plant Mol.Biol. 39:257-71. Additional endosperm specific promoters are disclosedin Albani, et al., (1984) EMBO 3:1405-15; Albani, et al., (1999) Theor.Appl. Gen. 98:1253-62; Albani, et al., (1993) Plant J. 4:343-55; Mena,et al., (1998) The Plant Journal 116:53-62, and Wu, et al., (1998) PlantCell Physiology 39:885-889.

Also of interest are promoters active in meristem regions, such asdeveloping inflorescence tissues, and promoters which drive expressionat or about the time of anthesis or early kernel development. This mayinclude, for example, the maize Zag promoters, including Zag1 and Zag2(see, Schmidt, et al., (1993) The Plant Cell 5:729-37; GenBank X80206;Theissen, et al., (1995) Gene 156:155-166; and U.S. patent applicationSer. No. 10/817,483); maize Zap promoter (also known as ZmMADS; U.S.patent application Ser. No. 10/387,937; WO 03/078590); maize ckx1-2promoter (U.S. patent publication 2002-0152500 A1; WO 02/0078438); maizeeep1 promoter (U.S. patent application Ser. No. 10/817,483); maize end2promoter (U.S. Pat. No. 6,528,704 and U.S. patent application Ser. No.10/310,191); maize lec1 promoter (U.S. patent application Ser. No.09/718,754); maize F3.7 promoter (Baszczynski, et al., Maydica42:189-201 (1997)); maize tb1 promoter (Hubbarda, et al., Genetics 162:1927-1935 (2002) and Wang, et al., (1999) Nature 398:236-239); maizeeep2 promoter (U.S. patent application Ser. No. 10/817,483); maizethioredoxinH promoter (U.S. provisional patent application 60/514,123);maize Zm40 promoter (U.S. Pat. No. 6,403,862 and WO 01/2178); maizemLIP15 promoter (U.S. Pat. No. 6,479,734); maize ESR promoter (U.S.patent application Ser. No. 10/786,679); maize PCNA2 promoter (U.S.patent application Ser. No. 10/388,359); maize cytokinin oxidasepromoters (U.S. patent application Ser. No. 11/094,917); promotersdisclosed in Weigal, et al., (1992) Cell 69:843-859; Accession No.AJ131822; Accession No. Z71981; Accession No. AF049870; andshoot-preferred promoters disclosed in McAvoy, et al., (2003) Acta Hort.(ISHS) 625:379-385. Other dividing cell or meristematic tissue-preferredpromoters that may be of interest have been disclosed in Ito, et al.,(1994) Plant Mol. Biol. 24:863-878; Regad, et al., (1995) Mo. Gen.Genet. 248:703-711; Shaul, et al., (1996) Proc. Natl. Acad. Sci.93:4868-4872; Ito, et al., (1997) Plant J. 11:983-992; and Trehin, etal., (1997) Plant Mol. Biol. 35:667-672, all of which are herebyincorporated by reference herein.

Inflorescence-preferred promoters include the promoter of chalconesynthase (Van der Meer, et al., (1990) Plant Mol. Biol. 15:95-109),LAT52 (Twell, et al., (1989) Mol. Gen. Genet. 217:240-245), pollenspecific genes (Albani, et al., (1990) Plant Mol. Biol. 15:605, Zm13(Buerrero, et al., (1993) Mol. Gen. Genet. 224:161-168), maizepollen-specific gene (Hamilton, et al., (1992) Plant Mol. Biol.18:211-218), sunflower pollen expressed gene (Baltz, et al., (1992) ThePlant Journal 2:713-721), and B. napus pollen specific genes (Arnoldo,et al., (1992) J. Cell. Biochem, Abstract No. Y101204).

Stress-inducible promoters include salt/water stress-inducible promoterssuch as P5CS (Zang, et al., (1997) Plant Sciences 129:81-89);cold-inducible promoters, such as, cor15a (Hajela, et al., (1990) PlantPhysiol. 93:1246-1252), cor15b (Wlihelm, et al., (1993) Plant Mol Biol23:1073-1077), wsc120 (Ouellet, et al., (1998) FEBS Lett. 423-324-328),ci7 (Kirch, et al., (1997) Plant Mol. Biol. 33:897-909), ci21A(Schneider, et al., (1997) Plant Physiol. 113:335-45); drought-induciblepromoters, such as, Trg-31 (Chaudhary, et al., (1996) Plant Mol. Biol.30:1247-57); osmotic inducible promoters, such as, Rab17 (Vilardell, etal., (1991) Plant Mol. Biol. 17:985-93) and osmotin (Raghothama, et al.,(1993) Plant Mol Biol 23:1117-28); and, heat inducible promoters, suchas, heat shock proteins (Barros, et al. (1992) Plant Mol. 19:665-75;Marrs, et al., (1993) Dev. Genet. 14:27-41), and smHSP (Waters, et al.,(1996) J. Experimental Botany 47:325-338). Other stress-induciblepromoters include rip2 (U.S. Pat. No. 5,332,808 and U.S. Publication No.2003/0217393) and rd29a (Yamaguchi-Shinozaki, et al., (1993) Mol. Gen.Genetics 236:331-340).

Stress-insensitive promoters can also be used in the methods of theinvention. This class of promoters, as well as representative examples,are further described elsewhere herein.

Nitrogen-responsive promoters can also be used in the methods of theinvention. Such promoters include, but are not limited to, the 22 kDaZein promoter (Spena, et al., (1982) EMBO J. 1:1589-1594 and Muller, etal., (1995) J. Plant Physiol 145:606-613); the 19 kDa zein promoter(Pedersen, et al., (1982) Cell 29:1019-1025); the 14 kDa zein promoter(Pedersen, et al., (1986) J. Biol. Chem. 261:6279-6284), the b-32promoter (Lohmer, et al., (1991) EMBO J 10:617-624); and the nitritereductase (NiR) promoter (Rastogi, et al., (1997) Plant Mol Biol.34(3):465-76 and Sander, et al., (1995) Plant Mol Biol. 27(1):165-77).For a review of consensus sequences found in nitrogen-induced promoters,see for example, Muller, et al., (1997) The Plant Journal 12:281-291.

Chemically-regulated promoters can be used to modulate the expression ofa gene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemically-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemically-induciblepromoters are known in the art and include, but are not limited to, themaize In2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237, and U.S.Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

A promoter induced by cytokinin, such as the ZmCkx1-2 promoter (U.S.Pat. No. 6,921,815, and pending U.S. patent application Ser. No.11/074,144), may also be used in the methods and compositions of theinvention. Such a construct would amplify biosynthesis of cytokininoccurring in developmental stages and/or tissues of interest. Othercytokinin-inducible promoters are described in pending U.S. patentapplication Ser. Nos. 11/094,917 and 11/273,537, all hereby incorporatedby reference.

Additional inducible promoters include heat shock promoters, such asGmhsp17.5-E (soybean) (Czarnecka, et al., (1989) Mol Cell Biol.9(8):3457-3463); APX1 gene promoter (Arabidopsis) (Storozhenko, et al.,(1998) Plant Physiol. 118(3):1005-1014): Ha hsp17.7 G4 (Helianthusannuus) (Almoguera, et al., (2002) Plant Physiol. 129(1):333-341; andMaize Hsp70 (Rochester, et al., (1986) EMBO J. 5: 451-8.

The methods of the invention involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the invention do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotides or polypeptides intoplants are known in the art and include, but are not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct of interest introduced into a plant integrates into the genomeof the plant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a sequence isintroduced into the plant and is only temporarily expressed or presentin the plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway, etal., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al.,(1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722),and ballistic particle acceleration (see, for example, U.S. Pat. No.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and, U.S.Pat. No. 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, andOrgan Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe, et al., (1988) Biotechnology6:923-926); and Lec1 transformation (WO 00/28058). Also see, Weissinger,et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987)Particulate Science and Technology 5:27-37 (onion); Christou, et al.,(1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Hoque, et al., (2005) Plant Cell Tissue & OrganCulture 82(1):45-55 (rice); Sreekala, et al., (2005) Plant Cell Reports24(2):86-94 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the IPT sequences of the invention can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the IPT protein or variants and fragments thereofdirectly into the plant or the introduction of an IPT transcript intothe plant. Such methods include, for example, microinjection or particlebombardment. See, for example, Crossway, et al., (1986) Mol. Gen. Genet.202:179-185; Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, et al.,(1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush, et al., (1994) TheJournal of Cell Science 107:775-784, all of which are hereinincorporated by reference. Alternatively, the IPT polynucleotide can betransiently transformed into the plant using techniques known in theart. Such techniques include viral vector system and the precipitationof the polynucleotide in a manner that precludes subsequent release ofthe DNA. Thus, the transcription from the particle-bound DNA can occur,but the frequency with which it is released to become integrated intothe genome is greatly reduced. Such methods include the use of particlescoated with polyethyenlimine (PEI; Sigma #P3143).

In other embodiments, the polynucleotide of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. It is recognized that an IPT polynucleotide of the inventionmay be initially synthesized as part of a viral polyprotein, which latermay be processed by proteolysis in vivo or in vitro to produce thedesired recombinant protein. Further, it is recognized that promotersuseful for the invention also encompass promoters utilized fortranscription by viral RNA polymerases. Methods for introducingpolynucleotides into plants and expressing a protein encoded therein,involving viral DNA or RNA molecules, are known in the art. See, forexample, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367,5,316,931, and Porta, et al., (1996) Molecular Biotechnology 5:209-221;herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, and U.S. Pat. Nos. 6,187,994; 6,552,248; 6,624,297;6,331,661; 6,262,341; 6,541,231; 6,664,108; 6,300,545; 6,528,700; and6,911,575, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in a transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andpollinated with either the same transformed strain or different strains,and the resulting progeny having expression of the desired phenotypiccharacteristic identified. Two or more generations may be grown toensure that expression of the desired phenotypic characteristic isstably maintained and inherited and then seeds harvested to ensure thatexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Pedigree breeding starts with the crossing of two genotypes, such as anelite line of interest and one other inbred line having one or moredesirable characteristics (i.e., having stably incorporated apolynucleotide of the invention, having a modulated activity and/orlevel of the polypeptide of the invention, etc) which complements theelite line of interest. If the two original parents do not provide allthe desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive filial generations. In the succeeding filialgenerations the heterozygous condition gives way to homogeneous lines asa result of self-pollination and selection. Typically in the pedigreemethod of breeding, five or more successive filial generations ofselfing and selection are practiced: F1→F2; F2→F3; F3 →F4; F4→F₅, etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed inbred. In specificembodiments, the inbred line comprises homozygous alleles at about 95%or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify anelite line of interest and a hybrid that is made using the modifiedelite line. Backcrossing can be used to transfer one or morespecifically desirable traits from one line, the donor parent, to aninbred called the recurrent parent, which has overall good agronomiccharacteristics yet lacks that desirable trait or traits. However, thesame procedure can be used to move the progeny toward the genotype ofthe recurrent parent but at the same time retain many components of thenon-recurrent parent by stopping the backcrossing at an early stage andproceeding with selfing and selection. For example, an F1, such as acommercial hybrid, is created. This commercial hybrid may be backcrossedto one of its parent lines to create a BC1 or BC2. Progeny are selfedand selected so that the newly developed inbred has many of theattributes of the recurrent parent and yet several of the desiredattributes of the non-recurrent parent. This approach leverages thevalue and strengths of the recurrent parent for use in new hybrids andbreeding.

Therefore, an embodiment of this invention is a method of making abackcross conversion of a maize inbred line of interest, comprising thesteps of crossing a plant of a maize inbred line of interest with adonor plant comprising a mutant gene or transgene conferring a desiredtrait (i.e., a modulation in the level of cytokinin (an increase or adecrease) or any plant phenotype resulting from the modulated cytokininlevel (such plant phenotypes are discussed elsewhere herein)), selectingan F1 progeny plant comprising the mutant gene or transgene conferringthe desired trait, and backcrossing the selected F1 progeny plant to aplant of the maize inbred line of interest. This method may furthercomprise the step of obtaining a molecular marker profile of the maizeinbred line of interest and using the molecular marker profile to selectfor a progeny plant with the desired trait and the molecular markerprofile of the inbred line of interest. In the same manner, this methodmay be used to produce F1 hybrid seed by adding a final step of crossingthe desired trait conversion of the maize inbred line of interest with adifferent maize plant to make F1 hybrid maize seed comprising a mutantgene or transgene conferring the desired trait.

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross pollinating with each other to form progeny. The progeny are grownand the superior progeny selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny,selfed progeny and topcrossing. The selected progeny arecross-pollinated with each other to form progeny for another population.This population is planted and again superior plants are selected tocross pollinate with each other. Recurrent selection is a cyclicalprocess and therefore can be repeated as many times as desired. Theobjective of recurrent selection is to improve the traits of apopulation. The improved population can then be used as a source ofbreeding material to obtain inbred lines to be used in hybrids or usedas parents for a synthetic cultivar. A synthetic cultivar is theresultant progeny formed by the intercrossing of several selectedinbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation breeding is one of many methods that could be used to introducenew traits into an elite line. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principals of Cultivar Development,” Fehr, 1993Macmillan Publishing Company, the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of elite lines that comprisessuch mutations.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn (Zeamays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularlythose Brassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsare optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as maize, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Typically, an intermediate host cell will be used in the practice ofthis invention to increase the copy number of the cloning vector. Withan increased copy number, the vector containing the nucleic acid ofinterest can be isolated in significant quantities for introduction intothe desired plant cells. In one embodiment, plant promoters that do notcause expression of the polypeptide in bacteria are employed.

Prokaryotes most frequently are represented by various strains of E.coli; however, other microbial strains may also be used. Commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding sequences, include such commonly usedpromoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan(trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res.8:4057) and the lambda derived P L promoter and N-gene ribosome bindingsite (Shimatake, et al., (1981) Nature 292:128). The inclusion ofselection markers in DNA vectors transfected in E. coli. is also useful.Examples of such markers include genes specifying resistance toampicillin, tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene22:229-235); Mosbach, et al., (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the presentinvention can be expressed in these eukaryotic systems. In someembodiments, transformed/transfected plant cells, as discussed infra,are employed as expression systems for production of the proteins of theinstant invention.

Synthesis of heterologous polynucleotides in yeast is well known(Sherman, et al., (1982) Methods in Yeast Genetics, Cold Spring HarborLaboratory). Two widely utilized yeasts for production of eukaryoticproteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors,strains, and protocols for expression in Saccharomyces and Pichia areknown in the art and available from commercial suppliers (e.g.,Invitrogen). Suitable vectors usually have expression control sequences,such as promoters, including 3-phosphoglycerate kinase or alcoholoxidase, and an origin of replication, termination sequences and thelike as desired. A protein of the present invention, once expressed, canbe isolated from yeast by lysing the cells and applying standard proteinisolation techniques to the lists. The monitoring of the purificationprocess can be accomplished by using Western blot techniques orradioimmunoassay or other standard immunoassay techniques.

The sequences of the present invention can also be ligated to variousexpression vectors for use in transfecting cell cultures of, forinstance, mammalian, insect, or plant origin. Illustrative cell culturesuseful for the production of the peptides are mammalian cells. A numberof suitable host cell lines capable of expressing intact proteins havebeen developed in the art, and include the HEK293, BHK21, and CHO celllines. Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter (e.g., the CMVpromoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter),an enhancer (Queen, et al., (1986) Immunol. Rev. 89:49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites (e.g., an SV40 large T Ag poly A additionsite), and transcriptional terminator sequences. Other animal cellsuseful for production of proteins of the present invention areavailable, for instance, from the American Type Culture Collection.

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See, Schneider(1987) J. Embryol. Exp. Morphol. 27:353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague, et al.,(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors (Saveria-Campo (1985)DNA Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press,Arlington, Va., pp. 213-238).

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextrin, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art (Kuchler(1997) Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc.).

II. Modulating the Concentration and/or Activity of an IsopentenylTransferase Polypeptide

A method for modulating the concentration and/or activity of thepolypeptide of the present invention in a plant is provided. In general,concentration and/or activity of the IPT polypeptide is increased orreduced by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or90% or more, relative to a native control plant, plant part, or cellwhich does not comprise the introduced sequence. Modulation of theconcentration and/or activity may occur at one or more stages ofdevelopment. In specific embodiments, the polypeptides of the presentinvention are modulated in monocots, such as maize.

The expression level of the IPT polypeptide may be measured directly,for example, by assaying for the level of the IPT polypeptide in theplant, or indirectly, for example, by measuring the cytokinin synthesisactivity in the plant. Methods for assaying for cytokinin synthesisactivity are described elsewhere herein.

In specific embodiments, the polypeptide or the polynucleotide of theinvention is introduced into the plant cell. Subsequently, a plant cellhaving the introduced sequence of the invention is selected usingmethods known to those of skill in the art such as, but not limited to,Southern blot analysis, DNA sequencing, PCR analysis, or phenotypicanalysis. A plant or plant part altered or modified by the foregoingembodiments is grown under plant forming conditions for a timesufficient to modulate the concentration and/or activity of polypeptidesof the present invention in the plant. Plant forming conditions are wellknown in the art and discussed briefly elsewhere herein.

It is also recognized that the level and/or activity of the polypeptidemay be modulated by employing a polynucleotide that is not capable ofdirecting, in a transformed plant, the expression of a protein or RNA.For example, the polynucleotides of the invention may be used to designpolynucleotide constructs that can be employed in methods for alteringor mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides, and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;all of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham, et al., (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference.

It is therefore recognized that methods of the present invention do notdepend on the incorporation of the entire polynucleotide into thegenome, only that the plant or cell thereof is altered as a result ofthe introduction of the polynucleotide into a cell. In one embodiment ofthe invention, the genome may be altered following the introduction of apolynucleotide into a cell. For example, the polynucleotide, or any partthereof, may incorporate into the genome of the plant. Alterations tothe genome include, but are not limited to, additions, deletions, andsubstitutions of nucleotides into the genome. While the methods of thepresent invention do not depend on additions, deletions, andsubstitutions of any particular number of nucleotides, it is recognizedthat such additions, deletions, or substitutions comprise at least onenucleotide.

It is further recognized that modulating the level and/or activity ofthe IPT sequence can be performed to elicit the effects of the sequenceonly during certain developmental stages and to switch the effect off inother stages where expression is no longer desirable. Control of the IPTexpression can be obtained via the use of inducible or tissue-preferredpromoters. Alternatively, the gene could be inverted or deleted usingsite-specific recombinases, transposons or recombination systems, whichwould also turn on or off expression of the IPT sequence.

A “subject plant or plant cell” is one in which genetic alteration, suchas transformation, has been effected as to a gene of interest, or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e. with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

In the present case, for example, changes in cytokinin levels, includingchanges in absolute amounts of cytokinin, cytokinin ratios, cytokininactivity, or cytokinin distribution, or changes in plant or plant cellphenotype, such as flowering time, seed set, branching, senescence,stress tolerance, or root mass, could be measured by comparing a subjectplant or plant cell to a control plant or plant cell.

In certain embodiments the nucleic acid constructs of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The polynucleotides of the present invention may bestacked with any gene or combination of genes, and the combinationsgenerated can include multiple copies of any one or more of thepolynucleotides of interest. The desired combination may affect one ormore traits; that is, certain combinations may be created for modulationof gene expression affecting cytokinin activity. For example,up-regulation of cytokinin synthesis may be combined withdown-regulation of cytokinin degradation. Other combinations may bedesigned to produce plants with a variety of desired traits, such asthose previously described.

A. Increasing the Activity and/or Concentration of an IsopentenylTransferase Polypeptide

Methods are provided to increase the activity and/or concentration ofthe IPT polypeptide of the invention. An increase in the concentrationand/or activity of the IPT polypeptide of the invention can be achievedby providing to the plant an IPT polypeptide. As discussed elsewhereherein, many methods are known in the art for providing a polypeptide toa plant including, but not limited to, direct introduction of thepolypeptide into the plant, and introducing into the plant (transientlyor stably) a polynucleotide construct encoding a polypeptide havingcytokinin synthesis activity. It is also recognized that the methods ofthe invention may employ a polynucleotide that is not capable ofdirecting, in the transformed plant, the expression of a protein or RNA.Thus, the level and/or activity of an IPT polypeptide may be increasedby altering the gene encoding the IPT polypeptide or its promoter. See,e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868.Therefore mutagenized plants that carry mutations in IPT genes, wherethe mutations increase expression of the IPT gene or increase thecytokinin synthesis activity of the encoded IPT polypeptide areprovided. As described elsewhere herein, methods to assay for anincrease in protein concentration or an increase in cytokinin synthesisactivity are known.

B. Reducing the Activity and/or Concentration of an IsopentenylTransferase Polypeptide

Methods are provided to reduce or eliminate the activity and/orconcentration of the IPT polypeptide by transforming a plant cell withan expression cassette that expresses a polynucleotide that inhibits theexpression of the IPT polypeptide. The polynucleotide may inhibit theexpression of an IPT polypeptide directly, by preventing translation ofthe IPT polypeptide messenger RNA, or indirectly, by encoding a moleculethat inhibits the transcription or translation of an IPT polypeptidegene encoding an IPT polypeptide. Methods for inhibiting or eliminatingthe expression of a gene in a plant are well known in the art, and anysuch method may be used in the present invention to inhibit theexpression of the IPT polypeptides.

In accordance with the present invention, the expression of an IPTpolypeptide is inhibited if the level of the IPT polypeptide isstatistically lower than the level of the same IPT polypeptide in aplant that has not been genetically modified or mutagenized to inhibitthe expression of that IPT polypeptide. In particular embodiments of theinvention, the protein level of the IPT polypeptide in a modified plantaccording to the invention is less than 95%, less than 90%, less than80%, less than 70%, less than 60%, less than 50%, less than 40%, lessthan 30%, less than 20%, less than 10%, or less than 5% of the proteinlevel of the same IPT polypeptide in a plant that is not a mutant orthat has not been genetically modified to inhibit the expression of thatIPT polypeptide. The expression level of the IPT polypeptide may bemeasured directly, for example, by assaying for the level of the IPTpolypeptide expressed in the cell or plant, or indirectly, for example,by measuring the cytokinin synthesis activity in the cell or plant.Methods for determining the cytokinin synthesis activity of the IPTpolypeptide are described elsewhere herein.

In other embodiments of the invention, the activity of one or more IPTpolypeptides is reduced or eliminated by transforming a plant cell withan expression cassette comprising a polynucleotide encoding apolypeptide that inhibits the activity of one or more IPT polypeptides.The cytokinin synthesis activity of an IPT polypeptide is inhibitedaccording to the present invention if the cytokinin synthesis activityof the IPT polypeptide is statistically lower than the cytokininsynthesis activity of the same IPT polypeptide in a plant that has notbeen genetically modified to inhibit the cytokinin synthesis activity ofthat IPT polypeptide. In particular embodiments of the invention, thecytokinin synthesis activity of the IPT polypeptide in a modified plantaccording to the invention is less than 95%, less than 90%, less than80%, less than 70%, less than 60%, less than 50%, less than 40%, lessthan 30%, less than 20%, less than 10%, or less than 5% of the cytokininsynthesis activity of the same IPT polypeptide in a plant that that hasnot been genetically modified to inhibit the expression of that IPTpolypeptide. The cytokinin synthesis activity of an IPT polypeptide is“eliminated” according to the invention when it is not detectable by theassay methods described elsewhere herein. Methods of determining thecytokinin synthesis activity of an IPT polypeptide are describedelsewhere herein.

In other embodiments, the activity of an IPT polypeptide may be reducedor eliminated by disrupting the gene encoding the IPT polypeptide. Theinvention encompasses mutagenized plants that carry mutations in IPTgenes, where the mutations reduce expression of the IPT gene or inhibitthe cytokinin synthesis activity of the encoded IPT polypeptide.

Thus, many methods may be used to reduce or eliminate the activity of anIPT polypeptide. More than one method may be used to reduce the activityof a single IPT polypeptide. In addition, combinations of methods may beemployed to reduce or eliminate the activity of two or more differentIPT polypeptides.

Non-limiting examples of methods of reducing or eliminating theexpression of an IPT polypeptide are given below.

1. Polynucleotide-Based Methods

In some embodiments of the present invention, a plant cell istransformed with an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of an IPT sequence. The term“expression” as used herein refers to the biosynthesis of a geneproduct, including the transcription and/or translation of said geneproduct. For example, for the purposes of the present invention, anexpression cassette capable of expressing a polynucleotide that inhibitsthe expression of at least one IPT sequence is an expression cassettecapable of producing an RNA molecule that inhibits the transcriptionand/or translation of at least one IPT polypeptide. The “expression” or“production” of a protein or polypeptide from a DNA molecule refers tothe transcription and translation of the coding sequence to produce theprotein or polypeptide, while the “expression” or “production” of aprotein or polypeptide from an RNA molecule refers to the translation ofthe RNA coding sequence to produce the protein or polypeptide.

Examples of polynucleotides that inhibit the expression of an IPTsequence are given below.

i. Sense Suppression/Cosuppression

In some embodiments of the invention, inhibition of the expression of anIPT polypeptide may be obtained by sense suppression or cosuppression.For cosuppression, an expression cassette is designed to express an RNAmolecule corresponding to all or part of a messenger RNA encoding an IPTpolypeptide in the “sense” orientation. Over expression of the RNAmolecule can result in reduced expression of the native gene.Accordingly, multiple plant lines transformed with the cosuppressionexpression cassette are screened to identify those that show thegreatest inhibition of IPT polypeptide expression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding the IPT polypeptide, all or part of the 5′and/or 3′ untranslated region of an IPT polypeptide transcript, or allor part of both the coding sequence and the untranslated regions of atranscript encoding an IPT polypeptide. In some embodiments where thepolynucleotide comprises all or part of the coding region for the IPTpolypeptide, the expression cassette is designed to eliminate the startcodon of the polynucleotide so that no protein product will betranscribed.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin, et al., (2002) PlantCell 14:1417-1432. Cosuppression may also be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit theexpression of endogenous genes in plants are described in Flavell, etal., (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Jorgensen, et al.,(1996) Plant Mol. Biol. 31:957-973; Johansen and Carrington (2001) PlantPhysiol. 126:930-938; Broin, et al., (2002) Plant Cell 14:1417-1432;Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Yu, et al.,(2003) Phytochemistry 63:753-763; and U.S. Pat. Nos. 5,034,323,5,283,184, and 5,942,657; each of which is herein incorporated byreference. The efficiency of cosuppression may be increased by includinga poly-dT region in the expression cassette at a position 3′ to thesense sequence and 5′ of the polyadenylation signal. See, U.S. PatentPublication No. 20020048814, herein incorporated by reference.Typically, such a nucleotide sequence has substantial sequence identityto the sequence of the transcript of the endogenous gene, optimallygreater than about 65% sequence identity, more optimally greater thanabout 85% sequence identity, most optimally greater than about 95%sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323, hereinincorporated by reference.

ii. Antisense Suppression

In some embodiments of the invention, inhibition of the expression ofthe IPT polypeptide may be obtained by antisense suppression. Forantisense suppression, the expression cassette is designed to express anRNA molecule complementary to all or part of a messenger RNA encodingthe IPT polypeptide. Over expression of the antisense RNA molecule canresult in reduced expression of the native gene. Accordingly, multipleplant lines transformed with the antisense suppression expressioncassette are screened to identify those that show the greatestinhibition of IPT polypeptide expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the IPTpolypeptide, all or part of the complement of the 5′ and/or 3′untranslated region of the IPT polypeptide transcript, or all or part ofthe complement of both the coding sequence and the untranslated regionsof a transcript encoding the IPT polypeptide. In addition, the antisensepolynucleotide may be fully complementary (i.e., 100% identical to thecomplement of the target sequence) or partially complementary (i.e.,less than 100% identical to the complement of the target sequence) tothe target sequence. Antisense suppression may be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, 300, 400, 450, 500, 550, or greater may be used. Methodsfor using antisense suppression to inhibit the expression of endogenousgenes in plants are described, for example, in Liu, et al., (2002) PlantPhysiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657, eachof which is herein incorporated by reference. Efficiency of antisensesuppression may be increased by including a poly-dT region in theexpression cassette at a position 3′ to the antisense sequence and 5′ ofthe polyadenylation signal. See, U.S. Patent Publication No.20020048814, herein incorporated by reference.

iii. Double-Stranded RNA Interference

In some embodiments of the invention, inhibition of the expression of anIPT polypeptide may be obtained by double-stranded RNA (dsRNA)interference. For dsRNA interference, a sense RNA molecule like thatdescribed above for cosuppression and an antisense RNA molecule that isfully or partially complementary to the sense RNA molecule are expressedin the same cell, resulting in inhibition of the expression of thecorresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence. Alternatively, separate expression cassettes maybe used for the sense and antisense sequences. Multiple plant linestransformed with the dsRNA interference expression cassette orexpression cassettes are then screened to identify plant lines that showthe greatest inhibition of IPT polypeptide expression. Methods for usingdsRNA interference to inhibit the expression of endogenous plant genesare described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA95:13959-13964, Liu, et al., (2002) Plant Physiol. 129:1732-1743, and WO99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of which isherein incorporated by reference.

iv. Hairpin RNA Interference and Intron-Containing Hairpin RNAInterference

In some embodiments of the invention, inhibition of the expression ofone or more IPT polypeptides may be obtained by hairpin RNA (hpRNA)interference or intron-containing hairpin RNA (ihpRNA) interference.These methods are highly efficient at inhibiting the expression ofendogenous genes. See, Waterhouse and Helliwell (2003) Nat. Rev. Genet.4:29-38 and the references cited therein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoding the gene whoseexpression is to be inhibited, and an antisense sequence that is fullyor partially complementary to the sense sequence. Thus, the base-pairedstem region of the molecule generally determines the specificity of theRNA interference. hpRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants. See, for example, Chuangand Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; andWaterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38. Methods forusing hpRNA interference to inhibit or silence the expression of genesare described, for example, in Chuang and Meyerowitz (2000) Proc. Natl.Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38;Pandolfini, et al., BMC Biotechnology 3:7, and U.S. Patent PublicationNo. 2003/0175965; each of which is herein incorporated by reference. Atransient assay for the efficiency of hpRNA constructs to silence geneexpression in vivo has been described by Panstruga, et al., (2003) Mol.Biol. Rep. 30:135-140, herein incorporated by reference.

Alternatively, the base-paired stem region may correspond to a portionof a promoter sequence controlling expression of the gene to beinhibited. Transcriptional gene silencing (TGS) may be accomplishedthrough use of hpRNA constructs wherein the inverted repeat of thehairpin shares sequence identity with the promoter region drivingexpression of a gene to be silenced. See, for example, U.S. patentapplication Ser. No. 11/014,071, filed 16 Dec. 2004. Processing of thehpRNA into short RNAs which can interact with the homologous promoterregion may trigger degradation or methylation to result in silencing(Aufsatz, et al., (2002) PNAS 99 (Suppl. 4):16499-16506; Mette, et al.,(2000) EMBO J 19(19):5194-5201).

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith, et al., (2000) Nature407:319-320. In fact, Smith, et al., show 100% suppression of endogenousgene expression using ihpRNA-mediated interference. Methods for usingihpRNA interference to inhibit the expression of endogenous plant genesare described, for example, in Smith, et al., (2000) Nature 407:319-320;Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse (2001)Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell (2003) Nat.Rev. Genet. 4:29-38; Helliwell and Waterhouse (2003) Methods 30:289-295,and U.S. Patent Publication No. 20030180945, each of which is hereinincorporated by reference.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, WO 02/00904, herein incorporated byreference.

v. Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for an IPT polypeptide). Methods ofusing amplicons to inhibit the expression of endogenous plant genes aredescribed, for example, in Angell and Baulcombe (1997) EMBO J.16:3675-3684, Angell and Baulcombe (1999) Plant J. 20:357-362, and U.S.Pat. No. 6,646,805, each of which is herein incorporated by reference.

vi. Ribozymes

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of an IPT polypeptide. Thus, thepolynucleotide causes the degradation of the endogenous messenger RNA,resulting in reduced expression of the IPT polypeptide. This method isdescribed, for example, in U.S. Pat. No. 4,987,071, herein incorporatedby reference.

vii. Small Interfering RNA or Micro RNA

In some embodiments of the invention, inhibition of the expression ofone or more IPT polypeptides may be obtained by RNA interference byexpression of a gene encoding a micro RNA (miRNA). miRNAs are regulatoryagents consisting of about 22 ribonucleotides. miRNA are highlyefficient at inhibiting the expression of endogenous genes. See, forexample, Javier, et al., (2003) Nature 425:257-263, herein incorporatedby reference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of IPT polypeptide expression, the22-nucleotide sequence is selected from an IPT polypeptide transcriptsequence and contains 22 nucleotides encoding said IPT polypeptidesequence in sense orientation and 21 nucleotides of a correspondingantisense sequence that is complementary to the sense sequence. miRNAmolecules are highly efficient at inhibiting the expression ofendogenous genes, and the RNA interference they induce is inherited bysubsequent generations of plants.

2. Polypeptide-Based Inhibition of Gene Expression

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding an IPT polypeptide, resulting in reducedexpression of the gene. In particular embodiments, the zinc fingerprotein binds to a regulatory region of an IPT polypeptide gene. Inother embodiments, the zinc finger protein binds to a messenger RNAencoding an IPT polypeptide and prevents its translation. Methods ofselecting sites for targeting by zinc finger proteins have beendescribed, for example, in U.S. Pat. No. 6,453,242, and methods forusing zinc finger proteins to inhibit the expression of genes in plantsare described, for example, in U.S. Patent Publication No. 20030037355;each of which is herein incorporated by reference.

3. Polypeptide-Based Inhibition of Protein Activity

In some embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one IPT polypeptide, and reduces thecytokinin synthesis activity of the IPT polypeptide. In anotherembodiment, the binding of the antibody results in increased turnover ofthe antibody-IPT polypeptide complex by cellular quality controlmechanisms. The expression of antibodies in plant cells and theinhibition of molecular pathways by expression and binding of antibodiesto proteins in plant cells are well known in the art. See, for example,Conrad and Sonnewald (2003) Nature Biotech. 21:35-36, incorporatedherein by reference.

4. Gene Disruption

In some embodiments of the present invention, the activity of an IPTpolypeptide is reduced or eliminated by disrupting the gene encoding theIPT polypeptide. The gene encoding the IPT polypeptide may be disruptedby any method known in the art. For example, in one embodiment, the geneis disrupted by transposon tagging. In another embodiment, the gene isdisrupted by mutagenizing plants using random or targeted mutagenesis,and selecting for plants that have reduced IPT activity.

i. Transposon Tagging

In one embodiment of the invention, transposon tagging is used to reduceor eliminate the cytokinin synthesis activity of one or more IPTpolypeptides. Transposon tagging comprises inserting a transposon withinan endogenous IPT gene to reduce or eliminate expression of the IPTpolypeptide. “IPT gene” is intended to mean the gene that encodes an IPTpolypeptide according to the invention.

In this embodiment, the expression of one or more IPT polypeptides isreduced or eliminated by inserting a transposon within a regulatoryregion or coding region of the gene encoding the IPT polypeptide. Atransposon that is within an exon, intron, 5′ or 3′ untranslatedsequence, a promoter, or any other regulatory sequence of an IPTpolypeptide gene may be used to reduce or eliminate the expressionand/or activity of the encoded IPT polypeptide.

Methods for the transposon tagging of specific genes in plants are wellknown in the art. See, for example, Maes, et al., (1999) Trends PlantSci. 4:90-96; Dharmapuri and Sonti (1999) FEMS Microbiol. Lett.179:53-59; Meissner, et al., (2000) Plant J. 22:265-274; Phogat, et al.,(2000) J. Biosci. 25:57-63; Walbot (2000) Curr. Opin. Plant Biol.2:103-107; Gai, et al., (2000) Nucleic Acids Res. 28:94-96; Fitzmaurice,et al., (1999) Genetics 153:1919-1928). In addition, the TUSC processfor selecting Mu insertions in selected genes has been described inBensen, et al., (1995) Plant Cell 7:75-84; Mena, et al., (1996) Science274:1537-1540; and U.S. Pat. No. 5,962,764; each of which is hereinincorporated by reference.

ii. Mutant Plants with Reduced Activity

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, such as ethyl methanesulfonate-inducedmutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesisused in a reverse genetics sense (with PCR) to identify plant lines inwhich the endogenous gene has been deleted. For examples of thesemethods see Ohshima, et al., (1998) Virology 243:472-481; Okubara, etal., (1994) Genetics 137:867-874; and Quesada, et al., (2000) Genetics154:421-436; each of which is herein incorporated by reference. Inaddition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products is also applicable to the instant invention. See, McCallum,et al., (2000) Nat. Biotechnol. 18:455-457, herein incorporated byreference.

Mutations that impact gene expression or that interfere with thefunction (IPT activity) of the encoded protein are well known in theart. Insertional mutations in gene exons usually result in null-mutants.Mutations in conserved residues are particularly effective in inhibitingthe cytokinin synthesis activity of the encoded protein. Conservedresidues of plant IPT polypeptides suitable for mutagenesis with thegoal to eliminate IPT activity have been described. See, for example,FIG. 1. Such mutants can be isolated according to well-known procedures,and mutations in different IPT loci can be stacked by genetic crossing.See, for example, Gruis, et al., (2002) Plant Cell 14:2863-2882.

In another embodiment of this invention, dominant mutants can be used totrigger RNA silencing due to gene inversion and recombination of aduplicated gene locus. See, for example, Kusaba, et al., (2003) PlantCell 15:1455-1467.

The invention encompasses additional methods for reducing or eliminatingthe activity of one or more IPT polypeptides. Examples of other methodsfor altering or mutating a genomic nucleotide sequence in a plant areknown in the art and include, but are not limited to, the use of RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides, and recombinogenic oligonucleobases. Such vectors andmethods of use are known in the art. See, for example, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;each of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham, et al., (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778; each of which is herein incorporatedby reference.

III. Modulating Cytokinin Level and/or Activity

As used herein, “cytokinin” refers to a class, or member of the class,of plant-specific hormones that play a central role during the cellcycle and influence numerous developmental programs. Cytokinins comprisean N⁶-substituted purine derivative. Representative cytokinins includeisopentenyladenine (N⁶-(Δ²-isopentenyl)adenine (hereinafter, iP), zeatin(6-(4-hydroxy-3-methylbut-trans-2-enylamino) purine) (hereinafter, Z),and dihydrozeatin (DZ). The free bases and their ribosides (iPR, ZR, andDZR) are believed to be the active compounds. Additional cytokinins areknown. See, for example, U.S. Pat. No. 5,211,738 and Keiber, et al.,(2002) Cytokinins, The Arabidopsis Book, American Society of PlantBiologists, both of which are herein incorporated by reference.

“Modulating the cytokinin level” includes any statistically significantdecrease or increase in cytokinin level and/or activity in the plantwhen compared to a control plant. For example, modulating the leveland/or activity can comprise either an increase or a decrease in overallcytokinin content of about 0.1%, 0.5%, 1%, 3% 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orgreater when compared to a control plant or plant part. Alternatively,the modulated level and/or activity of the cytokinin can include about a0.2 fold, 0.5 fold, 2 fold, 4 fold, 8 fold, 16 fold, 32 fold or greateroverall increase or decrease in cytokinin level/activity in the plant ora plant part when compared to a control plant or plant part.

It is further recognized that the modulation of the cytokininlevel/activity need not be an overall increase/decrease in cytokininlevel and/or activity, but also includes a change in tissue distributionof the cytokinin. Moreover, the modulation of the cytokininlevel/activity need not be an overall increase/decrease in cytokinins,but also includes a change in the ratio of various cytokininderivatives. For example, the ratio of various cytokinin derivativessuch as isopentenyladenine-type, zeatin-type, or dihydrozeatin-typecytokinins, and the like, could be altered and thereby modulate thelevel/activity of the cytokinin of the plant or plant part when comparedto a control plant.

Methods for assaying a modulation in cytokinin level and/or activity areknown in the art. For example, representative methods for cytokininextraction, immunopurification, HPLC separation, and quantification byELISA methods can be found, for example, in Faiss, et al., (1997) PlantJ. 12:401-415. See, also, Werner, et al., (2001) PNAS 98:10487-10492)and Dewitte, et al., (1999) Plant Physiol. 119:111-121. Each of thesereferences is herein incorporated by reference. As discussed elsewhereherein, modulation in cytokinin level and/or activity can further bedetected by monitoring for particular plant phenotypes. Such phenotypesare described elsewhere herein.

In specific methods, the level and/or activity of a cytokinin in a plantis increased by increasing the level or activity of the IPT polypeptidein the plant. Methods for increasing the level and/or activity of IPTpolypeptides in a plant are discussed elsewhere herein. Briefly, suchmethods comprise providing an IPT polypeptide of the invention to aplant and thereby increasing the level and/or activity of the IPTpolypeptide. In other embodiments, an IPT nucleotide sequence encodingan IPT polypeptide can be provided by introducing into the plant apolynucleotide comprising an IPT nucleotide sequence of the invention,expressing the IPT sequence, and thereby increasing the level and/oractivity of a cytokinin in the plant or plant part when compared to acontrol plant. In some embodiments, the IPT nucleotide constructintroduced into the plant is stably incorporated into the genome of theplant.

In other methods, the level and/or activity of cytokinin in a plant isdecreased by decreasing the level and/or activity of one or more of theIPT polypeptides in the plant. Such methods are disclosed in detailelsewhere herein. In one such method, an IPT nucleotide sequence isintroduced into the plant and expression of the IPT nucleotide sequencedecreases the activity of the IPT polypeptide, and thereby decreases thelevel and/or activity of a cytokinin in the plant or plant part whencompared to a control plant or plant part. In other embodiments, the IPTnucleotide construct introduced into the plant is stably incorporatedinto the genome of the plant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate the level/activity of a cytokinin in the plant.Exemplary promoters for this embodiment have been disclosed elsewhereherein.

Accordingly, the present invention further provides plants having amodulated level/activity of a cytokinin when compared to the cytokininlevel/activity of a control plant. In one embodiment, the plant of theinvention has an increased level/activity of the IPT polypeptide of theinvention, and thus has an increased level/activity of cytokinin. Inother embodiments, the plant of the invention has a reduced oreliminated level of the IPT polypeptide of the invention, and thus has adecreased level/activity of a cytokinin. In certain embodiments, suchplants have stably incorporated into their genome a nucleic acidmolecule comprising an IPT nucleotide sequence of the invention operablylinked to a promoter that drives expression in the plant cell.

IV. Modulating Root Development

Methods for modulating root development in a plant are provided. By“modulating root development” is intended any alteration in thedevelopment of the plant root when compared to a control plant. Suchalterations in root development include, but are not limited to,alterations in the growth rate of the primary root, the fresh rootweight, the extent of lateral and adventitious root formation, thevasculature system, meristem development, or radial expansion.

Methods for modulating root development in a plant are provided. Themethods comprise modulating the level and/or activity of the IPTpolypeptide in the plant. In one method, an IPT sequence of theinvention is provided to the plant. In another method, the IPTnucleotide sequence is provided by introducing into the plant apolynucleotide comprising an IPT nucleotide sequence of the invention(which may be a fragment of a full-length IPT sequence provided),expressing said IPT sequence, and thereby modifying root development. Instill other methods, the IPT nucleotide construct introduced into theplant is stably incorporated into the genome of the plant.

In other methods, root development is modulated by decreasing the levelor activity of the IPT polypeptide in the plant. Such methods cancomprise introducing an IPT nucleotide sequence into the plant anddecreasing the activity of the IPT polypeptide. In some methods, the IPTnucleotide construct introduced into the plant is stably incorporatedinto the genome of the plant. A decrease in cytokinin synthesis activitycan result in at least one or more of the following alterations to rootdevelopment, including, but not limited to, larger root meristems,increased root growth, enhanced radial expansion, an enhancedvasculature system, increased root branching, more adventitious roots,and/or an increase in fresh root weight when compared to a controlplant.

As used herein, “root growth” encompasses all aspects of growth of thedifferent parts that make up the root system at different stages of itsdevelopment in both monocotyledonous and dicotyledonous plants. It is tobe understood that enhanced root growth can result from enhanced growthof one or more of its parts including the primary root, lateral roots,adventitious roots, etc. Methods of measuring such developmentalalterations in the root system are known in the art. See, for example,U.S. Application No. 2003/0074698 and Werner, et al., (2001) PNAS18:10487-10492, both of which are herein incorporated by reference.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate root development in the plant. Exemplary promotersfor this embodiment include constitutive promoters and root-preferredpromoters. Exemplary root-preferred promoters have been disclosedelsewhere herein.

Stimulating root growth and increasing root mass by decreasing theactivity and/or level of the IPT polypeptide also finds use in improvingthe standability of a plant. The term “resistance to lodging” or“standability” refers to the ability of a plant to fix itself to thesoil. For plants with an erect or semi-erect growth habit, this termalso refers to the ability to maintain an upright position under adverseenvironmental conditions. This trait relates to the size, depth andmorphology of the root system. In addition, stimulating root growth andincreasing root mass by decreasing the level and/or activity of the IPTpolypeptide at appropriate developmental stages also finds use inpromoting in vitro propagation of explants.

Increased root biomass and/or altered root architecture may also finduse in improving nitrogen-use efficiency of the plant. Such improvedefficiency may lead to, for example, an increase in plant biomass and/orseed yield at an existing level of available nitrogen, or maintenance ofplant biomass and/or seed yield when available nitrogen is limited.Thus, agronomic and/or environmental benefits may ensue.

Furthermore, higher root biomass production due to a decreased leveland/or activity of an IPT polypeptide has an indirect effect onproduction of compounds produced by root cells or transgenic root cellsor cell cultures of said transgenic root cells. One example of aninteresting compound produced in root cultures is shikonin, the yield ofwhich can be advantageously enhanced by said methods.

Accordingly, the present invention further provides plants havingmodulated root development when compared to the root development of acontrol plant. In some embodiments, the plant of the invention has adecreased level/activity of an IPT polypeptide of the invention and hasenhanced root growth and/or root biomass. In certain embodiments, suchplants have stably incorporated into their genome a nucleic acidmolecule comprising an IPT nucleotide sequence of the invention operablylinked to a promoter that drives expression in the plant cell.

V. Modulating Shoot and Leaf Development

Methods are also provided for modulating vegetative tissue growth inplants. In one embodiment, shoot and leaf development in a plant ismodulated. By “modulating shoot and/or leaf development” is intended anyalteration in the development of the plant shoot and/or leaf whencompared to a control plant or plant part. Such alterations in shootand/or leaf development include, but are not limited to, alterations inshoot meristem development, in leaf number, leaf size, leaf and stemvasculature, internode length, and leaf senescence. As used herein,“leaf development” and “shoot development” encompasses all aspects ofgrowth of the different parts that make up the leaf system and the shootsystem, respectively, at different stages of their development, both inmonocotyledonous and dicotyledonous plants. Methods for measuring suchdevelopmental alterations in the shoot and leaf system are known in theart. See, for example, Werner, et al., (2001) PNAS 98:10487-10492 andU.S. Application No. 2003/0074698, each of which is herein incorporatedby reference.

The method for modulating shoot and/or leaf development in a plantcomprises modulating the activity and/or level of an IPT polypeptide ofthe invention. In one embodiment, an IPT sequence of the invention isprovided. In other embodiments, the IPT nucleotide sequence can beprovided by introducing into the plant a polynucleotide comprising anIPT nucleotide sequence of the invention, expressing the IPT sequence,and thereby modifying shoot and/or leaf development. In otherembodiments, the IPT nucleotide construct introduced into the plant isstably incorporated into the genome of the plant.

In specific embodiments, shoot or leaf development is modulated bydecreasing the level and/or activity of the IPT polypeptide in theplant. A decrease in IPT activity can result in one or more alterationsin shoot and/or leaf development, including, but not limited to, smallerapical meristems, reduced leaf number, reduced leaf surface, reducedvascular tissues, shorter internodes and stunted growth, and acceleratedleaf senescence, when compared to a control plant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate shoot and leaf development of the plant. Exemplarypromoters for this embodiment include constitutive promoters,shoot-preferred promoters, shoot meristem-preferred promoters,senescence-activated promoters, stress-induced promoters, root-preferredpromoters, nitrogen-induced promoters and leaf-preferred promoters.Exemplary promoters have been disclosed elsewhere herein.

Decreasing cytokinin synthesis activity in a plant generally results inshorter internodes and stunted growth. Thus, the methods of theinvention find use in producing dwarf plants. In addition, as discussedabove, modulation of cytokinin synthesis activity in the plant modulatesboth root and shoot growth. Thus, the present invention further providesmethods for altering the root/shoot ratio.

Shoot or leaf development can further be modulated by increasing thelevel and/or activity of the IPT polypeptide in the plant. An increasein IPT activity can result in one or more alterations in shoot and/orleaf development including, but not limited to, increased leaf number,increased leaf surface, increased vascular tissue, increased shootformation, longer internodes, improved growth, improved plant yield andvigor, and retarded leaf senescence when compared to a control plant.

In one embodiment, the tolerance of a plant to flooding is improved.Flooding is a serious environmental stress that affects plant growth andproductivity. Flooding causes premature senescence which results in leafchlorosis, necrosis, defoliation, cessation of growth and reduced yield.Cytokinins can regulate senescence, and by increasing the level/activityof the IPT polypeptide in the plant, the present invention improves thetolerance of the plant to a variety of environmental stresses, includingflooding. Delayed senescence may also advantageously expand the maturityadaptation of crops, improve the shelf-life of potted plants, and extendthe vase-life of cut flowers.

In still other embodiments, methods for modulating shoot regeneration ina callus are provided. In this method, increasing the level and/oractivity of the IPT polypeptide will increase the level of cytokinins inthe plant. Accordingly, lower concentrations of exogenous growthregulators (i.e., cytokinins) or no exogenous cytokinins in the culturemedium will be needed to enhance shoot regeneration in callus. Thus, inone embodiment of the invention, the increased level and/or activity ofthe IPT sequence can be used to overcome the poor shooting potential ofcertain species that has limited the success and speed of transgenetechnology for those species. Moreover, multiple shoot induction can beinduced for crops where it is economically desirable to produce as manyshoots as possible. Accordingly, methods are provided to increase therate of regeneration for transformation. In specific embodiments, theIPT sequence will be under the control of an inducible promoter (e.g.,heat shock promoter, chemically inducible promoter). Additionalinducible promoters are known in the art and are discussed elsewhereherein.

Methods for establishing callus from explants are known. For example,roots, stems, buds, and aseptically germinated seedlings are just a fewof the sources of tissue that can be used to induce callus formation.Generally, young and actively growing tissues (i.e., young leaves,roots, meristems or other tissues) are used, but are not required.Callus formation is controlled by growth regulating substances presentin the medium (auxins and cytokinins). The specific concentrations ofplant regulators needed to induce callus formation vary from species tospecies and can even depend on the source of explant. In some instances,it is advised to use different growth substances (e.g., 2, 4-D or NAA)or a combination of them during tests, since some species may notrespond to a specific growth regulator. In addition, culture conditions(i.e., light, temperature, etc.) can also influence the establishment ofcallus. Once established, callus cultures can be used to initiate shootregeneration. See, for example, Gurel, et al., (2001) Turk J. Bot.25:25-33; Dodds, et al., (1995). Experiments in Plant Tissue Culture,Cambridge University Press; Gamborg (1995) Plant Cell, Tissue and OrganCulture, eds. G. Phillips; and, U.S. Application No. 2003/0180952, allof which are herein incorporated by reference.

It is further recognized that increasing seed size and/or weight can beaccompanied by an increase in the rate of growth of seedlings or anincrease in vigor. In addition, modulating the plant's tolerance tostress, as discussed elsewhere herein, along with modulation of root,shoot and leaf development, can increase plant yield and vigor. As usedherein, the term “vigor” refers to the relative health, productivity,and rate of growth of the plant and/or of certain plant parts, and maybe reflected in various developmental attributes, including, but notlimited to, concentration of chlorophyll, photosynthetic rate, totalbiomass, root biomass, grain quality, and/or grain yield. In Zea mays inparticular, vigor may also be reflected in ear growth rate, ear size,and/or expansiveness of silk exsertion. Vigor may relate to the abilityof a plant to grow rapidly during early development and to thesuccessful establishment, after germination, of a well-developed rootsystem and a well-developed photosynthetic apparatus. Vigor may bedetermined with reference to different genotypes under similarenvironmental conditions, or with reference to the same or differentgenotypes under different environmental conditions.

Accordingly, the present invention further provides plants havingmodulated shoot and/or leaf development when compared to a controlplant. In some embodiments, the plant of the invention has an increasedlevel/activity of the IPT polypeptide of the invention. In otherembodiments, the plant of the invention has a decreased level/activityof the IPT polypeptide of the invention.

VI. Modulating Reproductive Tissue Development

Abortion of flowers and pods is a common occurrence in soybeans and isbelieved to limit yield (Abernethy, et al., (1997) Can J Plant Sci57:713-716; Dybing, et al., (1986) Plant Physiol 81:1069-1074).Cytokinins have been shown to play an important role during flower andpod development. Exogenous application of benzyladenine (a cytokinin) tothe raceme decreases abortion of flowers and/or pods (Dyer, et al.,(1988) In: Pharis and Rood, eds. Plant growth substances. New York:Springer-Verlag, 457-467; Peterson, et al., (1990) Botanical Gazette151:322-330; Mosjidis, et al., (1993) Annals of Botany 71:193-199;Reese, et al., (1995) J Exptl Botany 46(289):957-964) and a strong bodyof evidence supports a role for cytokinins in the regulation offlowering and seed setting in soybean (Huff and Dybing (1980) J ExptlBotany 31:51-762; Ghiasi, et al., (1987) Plant Physiol 81:1069-1074;Peterson, et al., (1990) Botanical Gazette 151:322-330; Wiebold (1990)Agron J 82:85-88; Mosjidis, et al., (1993), supra; Reese, et al.,(1995), supra; Nagel, et al., (2001) Annals of Botany 88:27-31).Increased number of pods and seed yields in response to cytokinintreatments support the hypothesis that increasing cytokininconcentration in developing flowers and pods using appropriate promoterswould result in increased total seed production of soybean plants.

Methods for modulating reproductive tissue development are provided. Inone embodiment, methods are provided to modulate floral development in aplant. By “modulating floral development” is intended any alteration ina structure of a plant's reproductive tissue as compared to a controlplant or plant part. “Modulating floral development” further includesany alteration in the timing of the development of a plant'sreproductive tissue (i.e., delayed or accelerated floral development)when compared to a control plant or plant part. Macroscopic alterationsmay include changes in size, shape, number, or location of reproductiveorgans, the developmental time period during which these structuresform, or the ability to maintain or proceed through the floweringprocess in times of environmental stress. Microscopic alterations mayinclude changes to the types or shapes of cells that make up thereproductive organs.

The method for modulating floral development in a plant comprisesmodulating (either increasing or decreasing) the level and/or activityof the IPT polypeptide in a plant. In one method, an IPT sequence of theinvention is provided. An IPT nucleotide sequence can be provided byintroducing into the plant a polynucleotide comprising an IPT nucleotidesequence of the invention, expressing the IPT sequence, and therebymodifying floral development. In some embodiments, the IPT nucleotideconstruct introduced into the plant is stably incorporated into thegenome of the plant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate floral development in the plant. Exemplary promotersfor this embodiment include constitutive promoters, inducible promoters,shoot-preferred promoters, and inflorescence-preferred promoters(including developing-female-inflorescence-preferred promoters),including those listed elsewhere herein.

In specific methods, floral development is modulated by increasing thelevel and/or activity of the IPT sequence of the invention. Such methodscan comprise introducing an IPT nucleotide sequence into the plant andincreasing the activity of the IPT polypeptide. In some methods, the IPTnucleotide construct introduced into the plant is stably incorporatedinto the genome of the plant. An increase in the level and/or activityof the IPT sequences can result in one or more alterations in floraldevelopment including, but not limited to, accelerated flowering,increased number of flowers, and improved seed set when compared to acontrol plant. In addition, an increase in the level or activity of theIPT sequences can result in the prevention of flower senescence and analteration in embryo number per kernel. See, Young, et al., (2004) PlantJ. 38:910-22. Methods for measuring such developmental alterations infloral development are known in the art. See, for example, Mouradov, etal., (2002) The Plant Cell S111-S130, herein incorporated by reference.

In other methods, floral development is modulated by decreasing thelevel and/or activity of the IPT sequence of the invention. A decreasein the level and/or activity of the IPT sequence can result in kernelabortion and infertile female inflorescence. Inducing delayed floweringor inhibiting flowering can be used to enhance yield in forage cropssuch as alfalfa.

Accordingly, the present invention further provides plants havingmodulated floral development when compared to the floral development ofa control plant. Compositions include plants having a decreasedlevel/activity of the IPT polypeptide of the invention and having analtered floral development. Compositions also include plants having anincreased level/activity of the IPT polypeptide of the invention whereinthe plant maintains or proceeds through the flowering process in timesof stress.

VII. Modulating the Stress Tolerance of a Plant

Methods are provided for the use of the IPT sequences of the inventionto modify the tolerance of a plant to abiotic stress. Increased growthof seedlings or early vigor is often associated with an increase instress tolerance. For example, faster development of seedlings,including the root system of seedlings upon germination, is critical forsurvival, particularly under adverse conditions such as drought.Promoters that can be used in this method are described elsewhereherein, including low-level constitutive, inducible, or root-preferredpromoters, such as root-preferred promoters derived from ZmIPT4 andZmIPT5 regulatory sequences. Accordingly, in one method of theinvention, a plant's tolerance to stress is increased or maintained whencompared to a control plant by decreasing the level of IPT activity inthe germinating seedling. In other methods, an IPT nucleotide sequenceis provided by introducing into the plant a polynucleotide comprising aIPT nucleotide sequence of the invention, expressing the IPT sequence,and thereby increasing the plant's tolerance to stress. In otherembodiments, the IPT nucleotide construct introduced into the plant isstably incorporated into the genome of the plant.

Methods are also provided to increase or maintain seed set duringabiotic stress episodes. During periods of stress (i.e., drought, salt,heavy metals, temperature, etc.) embryo development is often aborted. Inmaize, halted embryo development results in aborted kernels on the ear(Cheikh and Jones (1994) Plant Physiol. 106:45-51; Dietrich, et al.,(1995) Plant Physiol Biochem 33:327-336). In soy, abortion of pods priorto seed maturation can reduce seed yield and is observed during bothoptimal and stress conditions. Preventing this seed loss will maintainyield. Accordingly, methods are provided to increase the stressresistance in a plant (e.g., during flowering and seed development).Increasing expression of the IPT sequence of the invention can alsomodulate floral development during periods of stress, and thus methodsare provided to maintain or improve the flowering process in plantsunder stress. The method comprises increasing the level and/or activityof the IPT sequence of the invention. In one method, an IPT nucleotidesequence is introduced into the plant and the level and/or activity ofthe IPT polypeptide is increased, thereby maintaining or improving thetolerance of the plant under stress conditions. In other methods, theIPT nucleotide construct introduced into the plant is stablyincorporated into the genome of the plant. See, for example, WO00/63401.

Significant yield instability can occur as a result of unfavorableenvironments during the lag phase of seed development. During thisperiod, seeds undergo dramatic changes in ultra structure, biochemistry,and sensitivity to environmental perturbation, yet demonstrate littlechange in dry mass accumulation. Two important events that occur duringthe lag phase are initiation and division of endosperm cells andamyloplasts (which are the sites for starch deposition). It has beendemonstrated that during the lag phase (around 10-12 days afterpollination (DAP) in maize) a dramatic increase in cytokininconcentration immediately precedes maximum rates of endosperm celldivision and amyloplast formation, indicating that this hormone plays acentral role in these processes and in what is called the ‘sinkstrength’ of the developing seed. Cytokinins have been demonstrated toplay an important role in establishing seed size, decreasing seedabortion, and increasing seed set during unfavorable environmentalconditions. For example, elevated temperatures affect seed formation.Elevated temperatures can inhibit the accumulation of cytokinin,decrease endosperm cell division and amyloplast number, and as aconsequence, increase kernel abortion.

In crop species such as maize, kernel sink capacity is principally afunction of the number of endosperm cells and starch granulesestablished during the first 6 to 12 DAP. The final number of endospermcells and amyloplasts formed is highly correlated with final kernelweight. (Capitanio, et al., (1983); Reddy and Daynard, (1983); Jones, etal., (1985) (1996); Engelen-Eigles, et al., (2000)). Hormones,especially cytokinins, have been shown to stimulate cell division,plastid initiation and other processes important in the establishment ofkernel sink capacity (Davies, (1987)). Cytokinin levels could forexample be manipulated in soybean using the GmIPT2 promoter to drive theexpression of the Agrobacterium IPT gene. Similarly, endosperm- and/orpedicel-preferred promoters could be used to increase the level and/orduration of expression of GmIPT2, which would result in an increase ofcytokinin levels which would in turn increase flowers/pods retention,increasing sink strength and yield. Methods are therefore provided toincrease the activity and/or level of IPT polypeptides in the developinginflorescence, thereby elevating cytokinin levels and allowingdeveloping seed to achieve their full genetic potential for size,minimize pod and/or seed abortion, and buffer seed set duringunfavorable environments. The methods further allow the plant tomaintain and/or improve the flowering process during unfavorableenvironments.

In this embodiment, a variety of promoters could be used to direct theexpression of a sequence capable of increasing the level and/or activityof the IPT polypeptide, including but not limited to, constitutivepromoters, seed-preferred promoters, developing-seed promoters,meristem-preferred promoters, stress-induced promoters, andinflorescence-preferred (such as developing female inflorescencepromoters). In one method, a promoter that is stress insensitive and isexpressed in a tissue of the developing seed during the lag phase ofdevelopment is used. By “insensitive to stress” is intended that theexpression level of a sequence operably linked to the promoter is notaltered or only minimally altered under stress conditions. By “lagphase” promoter is intended a promoter that is active in the lag phaseof seed development. A description of this developmental phase is foundelsewhere herein. By “developing-seed-preferred” is intended a promoterthat allows for enhanced IPT expression within a developing seed. Suchpromoters that are stress insensitive and are expressed in a tissue ofthe developing seed during the lag phase of development are known in theart and include Zag2.1 (Theissen, et al., (1995) Gene 156:155-166,Genbank Accession No. X80206), and mzE40 (Zm40) (U.S. Pat. No. 6,403,862and WO01/2178).

An expression construct may further comprise nucleotide sequencesencoding peptide signal sequences in order to effect changes incytokinin level and/or activity in the mitochondria or chloroplasts.See, for example, Neupert (1997) Annual Rev. Biochem. 66:863-917;Glaser, et al., (1998) Plant Molecular Biology 38:311-338; Duby, et al.,(2001) The Plant J 27(6):539-549.

Methods to assay for an increase in seed set during abiotic stress areknown in the art. For example, plants having the increased IPT activitycan be monitored under various stress conditions and compared to controlplants. For instance, the plant having the increased cytokinin synthesisactivity can be subjected to various degrees of stress during floweringand seed set. Under identical conditions, the genetically modified planthaving the increased cytokinin synthesis activity will have a highernumber of developing pods and/or seeds than a control plant.

Accordingly, the present invention further provides plants havingincreased yield or a maintained yield and/or an increased or maintainedflowering process during periods of abiotic stress (drought, salt, heavymetals, temperature extremes, etc.). In some embodiments, the plantshaving an increased or maintained yield during abiotic stress have anincreased level/activity of the IPT polypeptide of the invention. Insome embodiments, the plant comprises an IPT nucleotide sequence of theinvention operably linked to a promoter that drives expression in theplant cell. In some embodiments, such plants have stably incorporatedinto their genome a nucleic acid molecule comprising an IPT nucleotidesequence of the invention operably linked to a promoter that drivesexpression in the plant cell.

VIII. Antibody Creation and Use

Antibodies can be raised to a protein of the present invention,including variants and fragments thereof, in both theirnaturally-occurring and recombinant forms. Many methods of makingantibodies are known to persons of skill. A variety of analytic methodsare available to generate a hydrophilicity profile of a protein of thepresent invention. Such methods can be used to guide the artisan in theselection of peptides of the present invention for use in the generationor selection of antibodies which are specifically reactive, underimmunogenic conditions, to a protein of the present invention. See,e.g., J. Janin, (1979) Nature, 277:491-492; Wolfenden, et al., (1981)Biochemistry 208:49-855; Kyte and Doolite, (1982) J. Mol. Biol.157:105-132; Rose, et al., (1985) Science 229:834-838. The antibodiescan be used to screen expression libraries for particular expressionproducts such as normal or abnormal protein, or altered levels of thesame, which may be useful for detecting or diagnosing various conditionsrelated to the presence of the respective antigens. Assays indicatinghigh levels of an IPT protein of the invention, for example, could beuseful in detecting plants, or specific plant parts, with elevatedcytokinin levels. Usually the antibodies in such a procedure are labeledwith a moiety which allows easy detection of presence ofantigen/antibody binding.

The following discussion is presented as a general overview of thetechniques available; however, one of skill will recognize that manyvariations upon the following methods are known.

A number of immunogens are used to produce antibodies specificallyreactive with a protein of the present invention. Polypeptides encodedby isolated recombinant, synthetic, or native polynucleotides of thepresent invention are the preferred antigens for the production ofmonoclonal or polyclonal antibodies. Polypeptides of the presentinvention are optionally denatured, and optionally reduced, prior toinjection into an animal capable of producing antibodies. Eithermonoclonal or polyclonal antibodies can be generated for subsequent usein immunoassays to measure the presence and quantity of the protein ofthe present invention. Methods of producing polyclonal antibodies areknown to those of skill in the art. In brief, an antigen, preferably apurified protein, a protein coupled to an appropriate carrier (e.g.,GST, keyhole limpet hemanocyanin, etc.), or a protein incorporated intoan immunization vector such as a recombinant vaccinia virus (see, U.S.Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunizedwith the mixture. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the protein of interest. When appropriately high titersof antibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Specific monoclonal and polyclonalantibodies will usually have an antibody binding site with an affinityconstant for its cognate monovalent antigen at least between 10⁶-10⁷,usually at least 10⁸, 10⁹, 10¹⁰ and up to about 10¹¹ liters/mole.Further fractionation of the antisera to enrich for antibodies reactiveto the protein is performed where desired (See, e.g., Coligan, (1991)Current Protocols in Immunology, Wiley/Greene, N.Y.; and Harlow andLane, (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor Press,N.Y.).

Antibodies, including binding fragments and single chain recombinantversions thereof, against predetermined fragments of a protein of thepresent invention are raised by immunizing animals, e.g., withconjugates of the fragments with carrier proteins as described above.Typically, the immunogen of interest is a protein of at least about 5amino acids, more typically the protein is 10 amino acids in length,often 15 to 20 amino acids in length, and may be longer. The peptidesare typically coupled to a carrier protein (e.g., as a fusion protein),or are recombinantly expressed in an immunization vector. Antigenicdeterminants on peptides to which antibodies bind are typically 3 to 10amino acids in length.

Monoclonal antibodies are prepared from hybrid cells secreting thedesired antibody. Monoclonal antibodies are screened for binding to aprotein from which the antigen was derived. Description of techniquesfor preparing such monoclonal antibodies are found in, e.g., Basic andClinical Immunology, 4th ed., Stites, et al., Eds., Lange MedicalPublications, Los Altos, Calif., and references cited therein; Harlowand Lane, Supra; Goding, Monoclonal Antibodies: Principles and Practice,2nd ed., Academic Press, New York, N.Y. (1986); and Kohler and Milstein,Nature 256:495-497 (1975). Summarized briefly, this method proceeds byinjecting an animal with an antigen comprising a protein of the presentinvention. The animal is then sacrificed and cells taken from itsspleen, which are fused with myeloma cells. The result is a hybrid cellor “hybridoma” that is capable of reproducing in vitro. The populationof hybridomas is then screened to isolate individual clones, each ofwhich secretes a single antibody species to the antigen. In this manner,the individual antibody species obtained are the products ofimmortalized and cloned single B cells generated by the animal inresponse to a specific site recognized on the antigenic substance.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors (see, e.g., Huse, et al., (1989)Science 246:1275-1281; and Ward, et al., (1989) Nature 341:544-546; andVaughan, et al., (1996) Nature Biotechnology, 14:309-314). Also,recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No.4,816,567; and Queen, et al. (1989), Proc. Nat'l Acad. Sci.86:10029-10033.

Antibodies to the polypeptides of the invention are also used foraffinity chromatography in isolating proteins of the present invention.Columns are prepared, e.g., with the antibodies linked to a solidsupport, e.g., particles, such as agarose, SEPHADEX, or the like, wherea cell lysate is passed through the column, washed, and treated withincreasing concentrations of a mild denaturant, whereby purifiedproteins are released.

Frequently, the proteins and antibodies of the present invention will belabeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like.

Protein Immunoassays

Means of detecting the proteins of the present invention are notcritical aspects of the present invention. In certain examples, theproteins are detected and/or quantified using any of a number ofwell-recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a general review ofimmunoassays, see also, Methods in Cell Biology, Vol. 37: Antibodies inCell Biology, Asai, Ed., Academic Press, Inc. New York (1993); Basic andClinical Immunology 7th Edition, Stites & Terr, Eds. (1991). Moreover,the immunoassays of the present invention can be performed in any ofseveral configurations, e.g., those reviewed in Enzyme Immunoassay,Maggio, Ed., CRC Press, Boca Raton, Fla. (1980); Tijan, Practice andTheory of Enzyme Immunoassays, Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers B.V., Amsterdam (1985);Harlow and Lane, supra; Immunoassay: A Practical Guide, Chan, Ed.,Academic Press, Orlando, Fla. (1987); Principles and Practice ofImmunoassays, Price and Newman Eds., Stockton Press, N.Y. (1991); andNon-isotopic Immunoassays, Ngo, Ed., Plenum Press, N.Y. (1988).

Immunological binding assays (or immunoassays) typically utilize a“capture agent” to specifically bind to and often immobilize the analyte(in this case, a protein of the present invention). The capture agent isa moiety that specifically binds to the analyte. In certain embodiments,the capture agent is an antibody that specifically binds a protein ofthe present invention. The antibody may be produced by any of a numberof means known to those of skill in the art as described herein.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledprotein of the present invention or a labeled antibody specificallyreactive to a protein of the present invention. Alternatively, thelabeling agent may be a third moiety, such as another antibody, thatspecifically binds to the antibody/protein complex.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, often from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

While the details of the immunoassays of the present invention may varywith the particular format employed, the method of detecting a proteinof the present invention in a biological sample generally comprises thesteps of contacting the biological sample with an antibody whichspecifically reacts, under immunologically reactive conditions, to aprotein of the present invention. The antibody is allowed to bind to theprotein under immunologically reactive conditions, and the presence ofthe bound antibody is detected directly or indirectly.

A. Non-Competitive Assay Formats

Immunoassays for detecting proteins of the present invention includecompetitive and noncompetitive formats. Noncompetitive immunoassays areassays in which the amount of captured analyte (i.e., a protein of thepresent invention) is directly measured. In one example, the “sandwich”assay, the capture agent (e.g., an antibody specifically reactive, underimmunoreactive conditions, to a protein of the present invention) can bebound directly to a solid substrate where it is immobilized. Theseimmobilized antibodies then capture the protein present in the testsample. The protein thus immobilized is then bound by a labeling agent,such as a second antibody bearing a label. Alternatively, the secondantibody may lack a label, but it may, in turn, be bound by a labeledthird antibody specific to antibodies of the species from which thesecond antibody is derived. The second antibody can be modified with adetectable moiety, such as biotin, to which a third labeled molecule canspecifically bind, such as enzyme-labeled streptavidin.

B. Competitive Assay Formats

In competitive assays, the amount of analyte present in the sample ismeasured indirectly by measuring the amount of an added (exogenous)analyte (e.g., a protein of the present invention) displaced (orcompeted away) from a capture agent (e.g., an antibody specificallyreactive, under immunoreactive conditions, to the protein) by theanalyte present in the sample. In one competitive assay, a known amountof analyte is added to the sample and the sample is then contacted witha capture agent that specifically binds a protein of the presentinvention. The amount of protein bound to the capture agent is inverselyproportional to the concentration of analyte present in the sample.

In one embodiment, the antibody is immobilized on a solid substrate. Theamount of protein bound to the antibody may be determined either bymeasuring the amount of protein present in a protein/antibody complex,or alternatively by measuring the amount of remaining uncomplexedprotein. The amount of protein may be detected by providing a labeledprotein.

A hapten inhibition assay is another competitive assay. In this assay aknown analyte, such as a protein of the present invention, isimmobilized on a solid substrate. A known amount of antibodyspecifically reactive, under immunoreactive conditions, to the proteinis added to the sample, and the sample is then contacted with theimmobilized protein. In this case, the amount of antibody bound to theimmobilized protein is inversely proportional to the amount of proteinpresent in the sample. Again, the amount of immobilized antibody may bedetermined by detecting either the immobilized fraction of antibody orthe fraction of the antibody that remains in solution. Detection may bedirect, where the antibody is labeled, or indirect, by the subsequentaddition of a labeled moiety that specifically binds to the antibody, asdescribed above.

C. Generation of Pooled Antisera for Use in Immunoassays

A protein that specifically binds to, or that is specificallyimmunoreactive with, an antibody generated against a defined antigen isdetermined in an immunoassay. The immunoassay uses a polyclonalantiserum which is raised to a polypeptide of the present invention(i.e., the antigenic polypeptide). This antiserum is selected to havelow cross-reactivity against other proteins, and any suchcross-reactivity is removed by immunoabsorption prior to use in theimmunoassay (e.g., by immunoabsorption of the antisera with a protein ofdifferent substrate specificity (e.g., a different enzyme) and/or aprotein with the same substrate specificity but of a different form).

In order to produce antisera for use in an immunoassay, a polypeptide ofthe present invention is isolated as described herein. For example,recombinant protein can be produced in a mammalian or other eukaryoticcell line. An inbred strain of mice is immunized with the protein usinga standard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, Harlow and Lane, supra). Alternatively, asynthetic polypeptide derived from the sequences disclosed herein andconjugated to a carrier protein is used as an immunogen. Polyclonal seraare collected and titered against the immunogenic polypeptide in animmunoassay, for example, a solid phase immunoassay with the immunogenimmobilized on a solid support. Polyclonal antisera with a titer of 10⁴or greater are selected and tested for their cross reactivity againstpolypeptides of different forms or substrate specificity, using acompetitive binding immunoassay such as the one described in Harlow andLane, supra, at pages 570-573. Preferably, two or more distinct forms ofpolypeptides are used in this determination. These distinct types ofpolypeptides are used as competitors to identify antibodies which arespecifically bound by the polypeptide being assayed for. The competitivepolypeptides can be produced as recombinant proteins and isolated usingstandard molecular biology and protein chemistry techniques as describedherein.

Immunoassays in the competitive binding format are used forcross-reactivity determinations. For example, the immunogenicpolypeptide is immobilized to a solid support. Proteins added to theassay compete with the binding of the antisera to the immobilizedantigen. The ability of the above proteins to compete with the bindingof the antisera to the immobilized protein is compared to theimmunogenic polypeptide. The percent cross-reactivity for the aboveproteins is calculated, using standard methods. Those antisera with lessthan 10% cross-reactivity for a distinct form of a polypeptide areselected and pooled. The cross-reacting antibodies are then removed fromthe pooled antisera by immunoabsorption with a distinct form of apolypeptide.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described herein to compare a second “target”polypeptide to the immunogenic polypeptide. In order to make thiscomparison, the two polypeptides are each assayed at a wide range ofconcentrations and the amount of each polypeptide required to inhibit50% of the binding of the antisera to the immobilized protein isdetermined using standard techniques. If the amount of the targetpolypeptide required is less than twice the amount of the immunogenicpolypeptide that is required, then the target polypeptide is said tospecifically bind to an antibody generated to the immunogenic protein.As a final determination of specificity, the pooled antisera is fullyimmunosorbed with the immunogenic polypeptide until no binding to thepolypeptide used in the immunoabsorption is detectable. The fullyimmunosorbed antisera is then tested for reactivity with the testpolypeptide. If no reactivity is observed, then the test polypeptide isspecifically bound by the antisera elicited by the immunogenic protein.

D. Other Assay Formats

In certain embodiments, Western blot (immunoblot) analysis is used todetect and quantify the presence of protein of the present invention inthe sample. The technique generally comprises separating sample proteinsby gel electrophoresis on the basis of molecular weight, transferringthe separated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind aprotein of the present invention. The antibodies specifically bind tothe protein on the solid support. These antibodies may be directlylabeled, or may be subsequently detected using labeled antibodies (e.g.,labeled sheep anti-mouse antibodies) that specifically bind to theantibodies.

E. Quantification of Proteins.

The proteins of the present invention may be detected and quantified byany of a number of means well known to those of skill in the art. Theseinclude analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,and various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like.

F. Reduction of Non-Specific Binding

One of skill will appreciate that it is often desirable to reducenon-specific binding in immunoassays and during analyte purification.Where the assay involves an antigen, antibody, or other capture agentimmobilized on a solid substrate, it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this involves coating the substrate with a proteinaceouscomposition. In particular, protein compositions such as bovine serumalbumin (BSA), nonfat powdered milk, and gelatin are widely used.

G. Immunoassay Labels

The labeling agent can be, e.g., a monoclonal antibody, a polyclonalantibody, a binding protein or complex, or a polymer such as an affinitymatrix, carbohydrate or lipid. Detectable labels suitable for use in thepresent invention include any composition detectable by spectroscopic,radioisotopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Detection may proceed by any known method,such as immunoblotting, Western analysis, gel-mobility shift assays,fluorescent in situ hybridization analysis (FISH), tracking ofradioactive or bioluminescent markers, nuclear magnetic resonance,electron paramagnetic resonance, stopped-flow spectroscopy, columnchromatography, capillary electrophoresis, or other methods which tracka molecule based upon an alteration in size and/or charge. Theparticular label or detectable group used in the assay is not a criticalaspect of the invention. The detectable group can be any material havinga detectable physical or chemical property, including magnetic beads,fluorescent dyes, radiolabels, enzymes, and colorimetric labels orcolored glass or plastic beads, as discussed for nucleic acid labels,supra. The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on the sensitivity required, ease of conjugation ofthe compound, stability requirements, available instrumentation, anddisposal provisions. Means of detecting labels are well known to thoseof skill in the art.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused.

The molecules can also be conjugated directly to signal-generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal-producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Assays for Compounds that Modulate Enzymatic Activity or Expression

A catalytically active polypeptide of the present invention may becontacted with a compound in order to determine whether said compoundbinds to and/or modulates the enzymatic activity of such polypeptide.The polypeptide employed will have at least 20%, 30%, 40%, 50%, 60%, 70%or 80% of the specific activity of the native, full-length enzyme of thepresent invention. Generally, the polypeptide will be present in a rangesufficient to determine the effect of the compound, typically about 1 nMto 10 μM. Likewise, the compound being tested will be present in aconcentration of from about 1 nM to 10 μM. Those of skill willunderstand that such factors as enzyme concentration, ligandconcentrations (i.e., substrates, products, inhibitors, activators), pH,ionic strength, and temperature will be controlled so as to obtainuseful kinetic data and determine the presence or absence of a compoundthat binds or modulates polypeptide activity. Methods of measuringenzyme kinetics are well known in the art. See, e.g., Segel, (1976)Biochemical Calculations, 2^(nd) ed., John Wiley and Sons, New York.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Cloning and Gene Characterization of GmIPT1 andGmIPT2

Below we describe the identification and characterization of two IPTpolypeptides from soybean (Glycine max) designated GmIPT1 and GmIPT2.

Material and methods: Sequences putatively representing IPT genes insoybean were initially identified by an in silico search of soy ESTdatabases using known Arabidopsis and maize IPT coding sequences. Twocandidate ESTs, pk0031 and pk086, were selected based on protein-levelhomology to the reference sequences and consideration of the libraryfrom which the candidate sequence originated.

Based on the candidate EST sequences, primers 100066, 100067, 100068,and 100069 (SEQ ID NOs: 10-13, respectively) were created. Primer pairs100066/100067 and 100068/100069 were used to screen a proprietarysoybean BAC library. Super-pools identified were further screened withprimer pair 100066/100067 and two BAC clones, CO₅ and 124, wereselected.

In each case, touchdown PCR was performed (GeneAmp® PCR System 9700,Applied Biosystems), using the following cycling parameters: 94° C. for3 min (one cycle), 94° C. for 1 min, 55° C. for 1 min and 72° C. for 1min 30 s, (35 cycles), 72° C. for 7 min, and termination at 4° C. PfuUltra Hotstar™ DNA polymerase (Stratagene) was used for its very lowaverage error rate (less than 0.5% per 500-bp fragment amplified).

Soybean insert DNA was isolated from the BAC clones and digested withEcoRI or PstI for Southern blot confirmation using the pk0031 EST cloneas a probe.

An EcoRI digestion of C05 was subcloned into pBluescript® (StratageneInc., La Jolla, Calif.). White colonies were grown in LB medium andtransferred onto a membrane using a dot-blot procedure. Afterdenaturation the membrane was probed with the pk0031 EST clone. Positiveclones were identified and sequenced.

FIG. 1 provides an amino acid alignment of the ZmIPT2, GmIPT1, GmIPT2,and GmIPT3 cytokinin biosynthetic enzymes. Asterisks indicate aminoacids conserved in many cytokinin biosynthetic enzymes. As shown in FIG.1, the deduced protein sequence of the GmIPT genes contains the exactconsensus sequenceGxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG[ST](SEQID NO: 9) (where x denotes any amino acid residue, [ ] any one of theamino acids shown in [ ], and x{m,n} m to n amino acid residues innumber) that was used by Takei, et al., (2001) J. Biol. Chem.276:26405-26410 to isolate the Arabidopsis ipt genes.

GAP-derived percentage sequence identity and sequence similarity valuesfor GmIPT 1, 2, and 3, relative to each other and to ZmIPT2 andArabidopsis IPT1-9, are shown in FIG. 2. Identity to other plant IPTproteins was found to be no higher than 52%.

Example 2 Expression of GmIPT Genes

In order to study the level of expression of the GmIPT genes in variousplant tissues, MPSS™ analysis (Solexa, Inc., Hayward, Calif.) wasperformed using 17-mer tags as shown in SEQ ID NOS: 14-16. In general,expression was found to be very low in most organs, but higher inreproductive tissues such as flowers (GmIPT1 and 2) and seed (GmIPT3).Tissue types, number of library hits, and average ppm for each arepresented in Table 1.

TABLE 1 Tissue Gene type Average ppm # of libraries GmIPT1 Flower 32.1 1GmIPT1 Leaf 3.5 2 GmIPT1 Stem 14.0 1 GmIPT1 Root 8.0 1 GmIPT2 Flower68.5 6 GmIPT2 Leaf 25.0 2 GmIPT2 Root 15.5 2 GmIPT2 Seed 13.2 5 GmIPT3Leaf 3.0 1 GmIPT3 Root 5.5 4 GmIPT3 Seed 13.3 12

Northern blots of GmIPT1 and GmIPT2 confirmed these findings. Theexpression pattern of GmIPT1 and GmIPT2 was further studied usingNorthern blot with RNA samples extracted from different soybean tissues(flowers, pods at different developmental stages, leaf, stem and root).GmIPT1 (AY550884) is expressed in stem and to a lesser extent in root,whereas GmIPT2 is highly expressed in roots and to a lesser extent insmall pods and stem. During pod development, GmIPT2 was found to beexpressed at higher levels in small pods and level of gene expressiondecreased as pod size and maturity increased. This suggests a moreimportant role of GmIPT2 in early stages of pod development.

Northern analysis of GmIPT3 expression is planned.

DNA and RNA extraction: Genomic DNA was extracted from plant samplesaccording to Dellaporta, et al., (1983) Plant Mol Biol 1:19-21 andstored at −20° C. Total RNA was prepared using a hot phenol extractionprocedure according to Verwoerd, et al., (1989) Nucleic Acid Res 17:2362and stored at −80° C. Samples were purified using RNEASY Mini Protocolfor RNA Cleanup (QIAgen) and eluted in 50 μl DEPC water. Optical Density(DO) at 260 and 280 nm was used to assess the purity of RNA preps andmeasure RNA and DNA concentrations.

Southern blots, Northern blots, and hybridization: For Southern blots,digested genomic or BAC clones DNA were run on 0.8% agarose gel at 110V,stained after migration in a 1:10000 (v/v) ethidium bromide solution inTAE buffer, and transferred as indicated below. For Northern blots,ethidium bromide was added to denatured RNA samples and run at 80 V on1.5% denaturing agarose gel (Brugiére, et al., (2003) Plant Physiol.132:1228-1240). Blotting was performed using Turbo-blotter (Schleicher &Schuell) according to the manufacturer guidelines. After transfer, nylonmembranes (Nytran plus, Schleicher & Schuell) were cross-linked with aStratalinker (Stratagene) and baked at 80° C. for 30 min. Probes werelabeled with [α-³²P]-dCTP using random priming (Rediprime II RandomPrimeLabelling System, Amersham Biosciences) and purified with Quick SpinColumns (Roche). Hybridizations were carried out at 65° C. for 16 husing ExpressHyb hybridization solution (BD Biosciences) and membraneswere washed under stringent conditions (0.1×SSC, 0.1% SDS) as previouslydescribed (Brugiére, et al., (2003) Plant Physiol. 132:1228-1240).Relative transcript abundance was quantified using a phosphor imager(MD860, Molecular Dynamic) with imaging software (ImageQuant, MolecularDynamics).

BAC subcloning: BAC clones were digested and subcloned in pBluescriptSK+. This plasmid includes a multiple cloning site between the lacZ geneand its promoter. The lacZ gene is often used as a reporter gene becauseit encodes a β-galactosidase, which produces a dark blue precipitate onX-gal enzymatic hydrolysis. The bacteria containing a plasmid in whichthe BAC fragment is inserted in the multiple cloning site and thereforedo not synthesize this enzyme will appear white. This allows theselection of colonies containing BAC subclones that can be furtherscreened by PCR or Southern blot.

Example 4 Maintaining or Increasing Seed Set During Stress

Targeted overexpression of the IPT sequences of the invention to thedeveloping female inflorescence of angiosperms, for example maize, soy,rice, or wheat, will elevate cytokinin levels and allow developing seedto achieve their full genetic potential for size, minimize seed and/orpod abortion, and buffer seed set during unfavorable environments.Abiotic stress that occurs during seed development in maize has beenshown to cause reduction in cytokinin levels. Under stress conditions,it is likely that cytokinin biosynthesis activity is decreased andcytokinin degradation is increased (Brugiére, et al., (2003) PlantPhysiol. 132(3):1228-40). Consequently, in one non-limiting method, tomaintain cytokinin levels in lag-phase seeds, IPT genes could be ligatedto control elements that: 1) are stress insensitive; 2) directexpression of structural genes predominantly to the developing seeds;and 3) preferentially drive expression of structural genes during thelag phase of seed development. Promoters which target expression torelated maternal tissues at or around anthesis may also be employed.Alternatively, a constitutive promoter could be employed.

Example 5 Maize Transformation

For example, immature maize embryos from greenhouse donor plants arebombarded with a plasmid containing a sequence, for example GmIPt2,operably linked to the Zag2.1 promoter (Schmidt, et al., (1993) PlantCell 5:729-737) and containing the selectable marker gene BAR(Wohlleben, et al., (1988) Gene 70:25-37), which confers resistance tothe herbicide Bialaphos. Alternatively, the selectable marker gene isprovided on a separate plasmid. Transformation is performed as follows.Media recipes follow below.

The ears are husked and surface-sterilized in 30% CLOROX bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5 cm target zone in preparation forbombardment.

A plasmid vector comprising the IPT sequence operably linked to a Zag2.1promoter is made. This plasmid DNA plus plasmid DNA containing a BARselectable marker is precipitated onto 1.1 μm (average diameter)tungsten pellets using a CaCl₂ precipitation procedure as follows: 100μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTAbuffer (1 μg total DNA); 100 μl 2.5 M CaCl₂; and, 10 μl 0.1 Mspermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for the maintenance or increase of seedset during an abiotic stress episode. In addition, transformants understress will be monitored for cytokinin levels (as described in Example5c) and maintenance of kernel growth.

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l GELRITE (added after bringing to volume with D-I H₂O); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000XSIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 3.0 g/l GELRITE (added after bringing to volume with D-I H₂O); and0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added aftersterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l GELRITE (addedafter bringing to volume with D-I H₂O); and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/lglycine brought to volume with polished D-I H₂O), 0.1 g/l myo-inositol,and 40.0 g/l sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6); and 6 g/l BACTO-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 6 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing the IPT sequenceoperably linked to a ubiquitin promoter as follows. To induce somaticembryos, cotyledons, 3-5 mm in length dissected from surface-sterilized,immature seeds of the soybean cultivar A2872, are cultured in the lightor dark at 26° C. on an appropriate agar medium for six to ten weeks.Somatic embryos producing secondary embryos are then excised and placedinto a suitable liquid medium. After repeated selection for clusters ofsomatic embryos that multiplied as early, globular-staged embryos, thesuspensions are maintained as described below.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 ml ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188), and the 3′ region of thenopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising the IPT sequenceoperably linked to the ubiquitin can be isolated as a restrictionfragment. This fragment can then be inserted into a unique restrictionsite of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Sunflower Meristem Tissue Transformation

Sunflower meristem tissues are transformed with an expression cassettecontaining the IPT sequence operably linked to a ubiquitin promoter asfollows (see also, European Patent Number EP 0 486233, hereinincorporated by reference, and Malone-Schoneberg, et al., (1994) PlantScience 103:199-207). Mature sunflower seed (Helianthus annuus L.) aredehulled using a single wheat-head thresher. Seeds are surfacesterilized for 30 minutes in a 20% CLOROX bleach solution with theaddition of two drops of TWEEN 20 per 50 ml of solution. The seeds arerinsed twice with sterile distilled water.

Split embryonic axis explants are prepared by a modification ofprocedures described by Schrammeijer, et al. (Schrammeijer, et al.,(1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled waterfor 60 minutes following the surface sterilization procedure. Thecotyledons of each seed are then broken off, producing a clean fractureat the plane of the embryonic axis. Following excision of the root tip,the explants are bisected longitudinally between the primordial leaves.The two halves are placed, cut surface up, on GBA medium consisting ofMurashige and Skoog mineral elements (Murashige, et al., (1962) Physiol.Plant. 15:473-497), Shepard's vitamin additions (Shepard (1980) inEmergent Techniques for the Genetic Improvement of Crops (University ofMinnesota Press, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/lsucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-aceticacid (IAA), 0.1 mg/l gibberellic acid (GA3), pH 5.6, and 8 g/l Phytagar.

The explants are subjected to microprojectile bombardment prior toAgrobacterium treatment (Bidney, et al., (1992) Plant Mol. Biol.18:301-313). Thirty to forty explants are placed in a circle at thecenter of a 60×20 mm plate for this treatment. Approximately 4.7 mg of1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TEbuffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are usedper bombardment. Each plate is bombarded twice through a 150 mm nytexscreen placed 2 cm above the samples in a PDS1000® particle accelerationdevice.

Disarmed Agrobacterium tumefaciens strain EHA105 is used in alltransformation experiments. A binary plasmid vector comprising theexpression cassette that contains the IPT gene operably linked to theubiquitin promoter is introduced into Agrobacterium strain EHA105 viafreeze-thawing as described by Holsters, et al., (1978) Mol. Gen. Genet.163:181-187. This plasmid further comprises a kanamycin selectablemarker gene (i.e., nptII). Bacteria for plant transformation experimentsare grown overnight (28° C. and 100 RPM continuous agitation) in liquidYEP medium (10 gm/l yeast extract, 10 gm/l Bactopeptone, and 5 gm/lNaCl, pH 7.0) with the appropriate antibiotics required for bacterialstrain and binary plasmid maintenance. The suspension is used when itreaches an OD₆₀₀ of about 0.4 to 0.8. The Agrobacterium cells arepelleted and resuspended at a final OD₆₀₀ of 0.5 in an inoculationmedium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH₄Cl, and 0.3 gm/lMgSO₄.

Freshly bombarded explants are placed in an Agrobacterium suspension,mixed, and left undisturbed for 30 minutes. The explants are thentransferred to GBA medium and co-cultivated, cut surface down, at 26° C.and 18-hour days. After three days of co-cultivation, the explants aretransferred to 374B (GBA medium lacking growth regulators and a reducedsucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants are cultured for two to five weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor one to two weeks of continued development. Explants withdifferentiating, antibiotic-resistant areas of growth that have notproduced shoots suitable for excision are transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of NPTII by ELISA and for the presence oftransgene expression by assaying for cytokinin synthesis activity. Suchassays are described elsewhere herein.

NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3%GELRITE, pH 5.6) and grown under conditions described for explantculture. The upper portion of the seedling is removed, a 1 cm verticalslice is made in the hypocotyl, and the transformed shoot inserted intothe cut. The entire area is wrapped with parafilm to secure the shoot.Grafted plants can be transferred to soil following one week of in vitroculture. Grafts in soil are maintained under high humidity conditionsfollowed by a slow acclimatization to the greenhouse environment.Transformed sectors of T₀ plants (parental generation) maturing in thegreenhouse are identified by NPTII ELISA and/or by cytokinin synthesisactivity analysis of leaf extracts while transgenic seeds harvested fromNPTII-positive T₀ plants are identified by cytokinin synthesis activityanalysis of small portions of dry seed cotyledon.

Example 8 Rice Transformation

One method for transforming DNA into cells of higher plants that isavailable to those skilled in the art is high-velocity ballisticbombardment using metal particles coated with the nucleic acidconstructs of interest (see, Klein, et al., Nature (1987) (London)327:70-73, and see, U.S. Pat. No. 4,945,050). A Biolistic PDS-1000/He(BioRAD Laboratories, Hercules, Calif.) is used for thesecomplementation experiments.

The bacterial hygromycin B phosphotransferase (Hpt II) gene fromStreptomyces hygroscopicus that confers resistance to the antibiotic maybe used as the selectable marker for rice transformation. In the vector,the Hpt II gene may be engineered with the 35S promoter from CauliflowerMosaic Virus and the termination and polyadenylation signals from theoctopine synthase gene of Agrobacterium tumefaciens. For example, seethe description of vector pML18 in WO 97/47731, published on Dec. 18,1997, the disclosure of which is hereby incorporated by reference.

Embryogenic callus cultures derived from the scutellum of germinatingrice seeds serve as source material for transformation experiments. Thismaterial is generated by germinating sterile rice seeds on a callusinitiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-Dand 10 μM AgNO₃) in the dark at 27-28° C. Embryogenic callusproliferating from the scutellum of the embryos is transferred to CMmedia (N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D, Chu, et al.,(1985) Sci. Sinica 18:659-668). Callus cultures are maintained on CM byroutine sub-culture at two-week intervals and used for transformationwithin 10 weeks of initiation.

Callus is prepared for transformation by subculturing 0.5-1.0 mm piecesapproximately 1 mm apart, arranged in a circular area of about 4 cm indiameter, in the center of a circle of Whatman #541 paper placed on CMmedia. The plates with callus are incubated in the dark at 27-28° C. for3-5 days. Prior to bombardment, the filters with callus are transferredto CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr inthe dark. The petri dish lids are then left ajar for 20-45 minutes in asterile hood to allow moisture on tissue to dissipate.

Each genomic DNA fragment is co-precipitated with pML18 (containing theselectable marker for rice transformation) onto the surface of goldparticles. To accomplish this, a total of 10 μg of DNA at a 2:1 ratio oftrait:selectable marker DNAs are added to 50 μl aliquot of goldparticles that have been resuspended at a concentration of 60 mg ml⁻¹.Calcium chloride (50 μl of a 2.5 M solution) and spermidine (20 μl of a0.1 M solution) are then added to the gold-DNA suspension as the tube isvortexing for 3 min. The gold particles are centrifuged in a microfugefor 1 sec and the supernatant removed. The gold particles are washedtwice with 1 ml of absolute ethanol and then resuspended in 50 μl ofabsolute ethanol and sonicated (bath sonicator) for one second todisperse the gold particles. The gold suspension is incubated at −70° C.for five minutes and sonicated (bath sonicator) if needed to dispersethe particles. Six μl of the DNA-coated gold particles are then loadedonto mylar macrocarrier disks and the ethanol is allowed to evaporate.

At the end of the drying period, a petri dish containing the tissue isplaced in the chamber of the PDS-1000/He. The air in the chamber is thenevacuated to a vacuum of 28-29 inches Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1080-1100 psi. Thetissue is placed approximately 8 cm from the stopping screen and thecallus is bombarded two times. Two to four plates of tissue arebombarded in this way with the DNA-coated gold particles. Followingbombardment, the callus tissue is transferred to CM media withoutsupplemental sorbitol or mannitol.

Within 3-5 days after bombardment the callus tissue is transferred to SMmedia (CM medium containing 50 mg/l hygromycin). To accomplish this,callus tissue is transferred from plates to sterile 50 ml conical tubesand weighed. Molten top-agar at 40° C. is added using 2.5 ml of topagar/100 mg of callus. Callus clumps are broken into fragments of lessthan 2 mm diameter by repeated dispensing through a 10 ml pipet. Threeml aliquots of the callus suspension are plated onto fresh SM media andthe plates are incubated in the dark for 4 weeks at 27-28° C. After 4weeks, transgenic callus events are identified, transferred to fresh SMplates and grown for an additional 2 weeks in the dark at 27-28° C.

Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitschvitamins, 2% sucrose, 3% sorbitol, 0.4% GELRITE+50 ppm hyg B) for 2weeks in the dark at 25° C. After 2 weeks the callus is transferred toRM2 media (MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4%GELRITE+50 ppm hyg B) and placed under cool white light (˜40 μEm⁻²s⁻¹)with a 12 hr photoperiod at 25° C. and 30-40% humidity. After 2-4 weeksin the light, callus begin to organize, and form shoots. Shoots areremoved from surrounding callus/media and gently transferred to RM3media (1/2×MS salts, Nitsch and Nitsch vitamins, 1% sucrose+50 ppmhygromycin B) in phytatrays (Sigma Chemical Co., St. Louis, Mo.) andincubation is continued using the same conditions as described in theprevious step.

Plants are transferred from RM3 to 4″ pots containing Metro mix 350after 2-3 weeks, when sufficient root and shoot growth have occurred.

Example 9 Modulating Root Development

For Agrobacterium-mediated transformation of soybean with a plasmiddesigned to achieve post-transcriptional gene silencing (PTGS) with anappropriate promoter, the method of Zhao may be employed (U.S. Pat. No.5,981,840, and PCT patent publication WO98/32326, the contents of whichare hereby incorporated by reference). Briefly, immature embryos areisolated and contacted with a suspension of Agrobacterium capable oftransferring a DNA construct. Said construct may comprise the CRWAQ81root-preferred promoter::ADH intron promoter operably linked to ahairpin structure made from the coding sequence of any one of the GmIPTpolynucleotides of the invention. Other useful constructs may comprise ahairpin construct targeting the promoter of any one of the GmIPTpolynucleotides of the invention. (Aufsatz, et al., (2002) PNAS99(4):16499-16506; Mette, et al., (2000) EMBO J 19(19):5194-5201) Theconstruct is transferred to at least one cell of at least one of theimmature embryos (step 1: the infection step). In this step the immatureembryos are immersed in an Agrobacterium suspension for the initiationof inoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step); this may take place onsolid medium. Following this co-cultivation period an optional “resting”step is contemplated. In this resting step, the embryos are incubated inthe presence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Next, inoculated embryos arecultured on medium containing a selective agent; growing, transformedcallus is recovered (step 4: the selection step). The callus is thenregenerated into plants (step 5: the regeneration step).

Plants are monitored and scored for a modulation in root development.The modulation in root development includes monitoring for enhanced rootgrowth of one or more root parts including the primary root, lateralroots, adventitious roots, etc. Methods of measuring such developmentalalterations in the root system are known in the art. See, for example,U.S. Application No. 2003/0074698 and Werner, et al., (2001) PNAS18:10487-10492, both of which are herein incorporated by reference.

Example 10 Modulating Senescence of a Plant

A DNA construct comprising the GmIPT1 or GmIPT2 polynucleotide operablylinked to a constitutive promoter, a root-preferred promoter, or asenescence-activated promoter, such as SAG12 (Gan, et al., (1995)Science 270:5244, Genbank Acc. No. U37336) is introduced into maizeplants as outlined in Zhao, et al., (1998) Maize Genetics CorporationNewsletter 72:34-37, herein incorporated by reference.

For example, maize plants comprising an IPT sequence operably linked tothe SAG12 promoter are obtained. As a control, a non-cytokinin-relatedconstruct is also introduced into maize plants using the transformationmethod outlined above. The phenotypes of transgenic maize plants havingan elevated level of the IPT polypeptide are studied. For example,plants can be monitored for an improved vitality, shelf and vase life,and improved tolerance against infection. Plants could also be monitoredfor delayed senescence under various environmental stresses including,for example, flooding which normally results in leaf chlorosis,necrosis, defoliation, cessation of growth and reduction in yield.

Example 11 Variants of IPT

A. Variant Nucleotide Sequences of GmIPT1, GmIPT2, or GmIPT3 that Do NotAlter the Encoded Amino Acid Sequence

The GmIPT nucleotide sequences set forth in SEQ ID NO: 1, 3 and 6 areused to generate variant nucleotide sequences having the nucleotidesequence of the open reading frame with about 70%, 75%, 80%, 85%, 90% or95% nucleotide sequence identity when compared to the correspondingstarting unaltered ORF nucleotide sequence. These functional variantsare generated using a standard codon table. While the nucleotidesequence of the variant is altered, the amino acid sequence encoded bythe open reading frame does not change.

B. Variant Amino Acid Sequences of GmIPT1, GmIPT2, and GmIPT3

Variant amino acid sequences of GmIPT1, GmIPT2 and GmIPT3 are generated.In this example, one or more amino acids are altered. Specifically, theopen reading frame set forth in SEQ ID NO: 2, 4 or 7 is reviewed todetermine the appropriate amino acid alteration. The selection of anamino acid to change is made by consulting a protein alignment withorthologs and other gene family members from various species. See, FIG.1 and/or FIG. 4. An amino acid is selected that is deemed not to beunder high selection pressure (not highly conserved) and which is rathereasily substituted by an amino acid with similar chemicalcharacteristics (i.e., similar functional side-chain). Assays asoutlined elsewhere herein may be followed to confirm functionality.Variants having about 70%, 75%, 80%, 85%, 90% or 95% nucleic acidsequence identity to each of SEQ ID NO: 2, 4 and 7 are generated usingthis method.

C. Additional Variant Amino Acid Sequences of GmIPT1 and GmIPT2

In this example, artificial protein sequences are created having 80%,85%, 90% and 95% identity relative to the reference protein sequence.This latter effort requires identifying conserved and variable regionsfrom the alignment set forth in FIG. 1 and then the judiciousapplication of an amino acid substitutions table. These parts will bediscussed in more detail below.

Largely, the determination of which amino acid sequences are altered ismade based on the conserved regions among the IPT proteins or among theother IPT polypeptides. See FIG. 1. Based on the sequence alignment, thevarious regions of the IPT polypeptides that can likely be altered canbe determined. It is recognized that conservative substitutions can bemade in the conserved regions without altering function. In addition,one of skill will understand that functional variants of the IPTsequence of the invention can have minor non-conserved amino acidalterations in the conserved domain.

Artificial protein sequences are then created that are different fromthe original in the intervals of 80-85%, 85-90%, 90-95% and 95-100%identity. Midpoints of these intervals are targeted, with liberallatitude of plus or minus 1%, for example. The amino acids substitutionswill be effected by a custom Perl script. The substitution table isprovided below in Table 2.

First, any conserved amino acids in the protein that should not bechanged are identified and “marked off” for insulation from thesubstitution. The start methionine will of course be added to this listautomatically. Next, the changes are made.

H, C and P are not changed. The changes will occur with isoleucinefirst, sweeping N-terminal to C-terminal. Then leucine, and so on downthe list until the desired target is reached. Interim numbersubstitutions can be made so as not to cause reversal of changes. Thelist is ordered 1-17, so start with as many isoleucine changes as neededbefore leucine, and so on down to methionine. Clearly many amino acidswill in this manner not need to be changed. L, I and V will involve a50:50 substitution of the two alternate optimal substitutions.

The variant amino acid sequences are written as output. Perl script isused to calculate the percent identities. Using this procedure, variantsof GmIPT1 and GmIPT2 are generated having about 82%, 87%, 92%, and 97%amino acid identity to the starting unaltered ORF nucleotide sequence ofSEQ ID NO: 2 or 4.

TABLE 2 Substitution Table Strongly Rank of Similar and Order AminoOptimal to Acid Substitution Change Comment I L, V 1 50:50 substitutionL I, V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E6 E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L17 First methionine cannot change H Na No good substitutes C Na No goodsubstitutes P Na No good substitutes

Example 12 Amplification of Additional Isopentenyl Transferase (IPT)Genes from Soybean or Other Plant Species

Additional IPT genes from plant species could be identified by PCR orRT-PCR methods using degenerate primers such as the ones describedbelow. Degenerate primers can be designed against conserved amino acidmotifs found in available IPT proteins from soybean, maize, rice orArabidopsis. Such motifs can be identified from an alignment of theprotein sequences. Examples of sequences of such motifs andcorresponding degenerate nucleotide primers are listed below:

Amino Sense degenerate Antisense acid motif primer degenerate primerEIINSDK(I/M)Q GAR ATH ATH AAY TG IAT YTT RTC ISW SEQ ID NO: 17 WSI GAYAAR ATI RTT DAT DAT YTC CA SEQ ID NO: 18 SEQ ID NO: 19 GVPHHLLG GGI GTICCI CAY CC IAR IAR RTG RTG SEQ ID NO: 20 CAY YTI YTI GG IGG IAC ICC SEQID NO: 21 SEQ ID NO: 22 GVPHHLL GGI GTI CCI CAY IAR IAR RTG RTG SEQ IDNO: 23 CAY YTI YT IGG IAC ICC SEQ ID NO: 24 SEQ ID NO: 25 AGGSN GCI GGIGGI WSI RTT ISW ICC ICC SEQ ID NO: 26 AAY SEQ ID NO: 27 IGC SEQ ID NO:28 (A/V)GGSNS(Y/F) GYI GGI GGI WSI RWA ISW RTT ISW SEQ ID NO: 29 AAY WSITWY ICC ICC IRC SEQ ID NO: 30 SEQ ID NO: 31 CCF[I/L]WVDV TGY TGY TTY HTIAC RTC IAC CCA IAD SEQ ID NO: 32 TGG GTI GAY GT RAA RCA RCA SEQ ID NO:33 SEQ ID NO: 34

Sense/antisense primers could be used in different combinations.Similarly, several rounds of PCR could be used. The product ofamplification of one pair of sense/antisense primers could be used astemplate for PCR with another set of internal (nested) degenerateprimers therefore maximizing the chances for amplification of anappropriate sequence, i.e., containing a sequence corresponding to thecorresponding amino acid motif.

Nucleotide Symbols A A Adenine C C Cytosine G G Guanine T T Thymine U UUracil I I Inosine R A or G puRine Y C or T (U) pYrimidine M A or CaMino K G or T (U) Keto S C or G Strong (triple ‘3 H’ bonds) W A or T(U) Weak (double ‘2 H’ bonds) B C or G or T (U) not A D A or G or T (U)not C H A or C or T (U) not G V A or C or G not T (U) N A or C or G or T(U) aNy nucleotide

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated or recombinant polynucleotide comprising the completenucleotide sequence of SEQ ID NO:
 3. 2. A transgenic plant or plant partcomprising an isolated or recombinant polynucleotide operably linked toa promoter that drives expression in the plant or plant part, whereinsaid polynucleotide comprises the complete nucleotide sequence of SEQ IDNO:
 3. 3. The plant of claim 2, wherein said polynucleotide is operablylinked to a tissue-preferred promoter, a constitutive promoter, or aninducible promoter.
 4. The plant of claim 3, wherein saidtissue-preferred promoter is a root-preferred promoter, a leaf-preferredpromoter, a shoot-preferred promoter, or an inflorescence-preferredpromoter.
 5. The plant of claim 2, wherein said promoter isstress-insensitive and is expressed in a tissue of the developing seedor related maternal tissue at or about the time of anthesis.
 6. Theplant part of claim 2, wherein said part is a transformed seed.
 7. Anisolated or recombinant polynucleotide which encodes a polypeptide whichis 95% identical to the full length of SEQ ID NO: 4 and which comprisesthe consensus sequence of SEQ ID NO: 9.